Questionnaire/Script Form 74 before AV NBS Telephone ... · Participant Materials and Appendix D...

NBS Manual

WHI NUTRITIONAL BIOMARKERS STUDY (NBS) VER 1: MAY 2004

SECTION 1

WHI NUTRITIONAL BIOMARKERS STUDY OVERVIEW

1.0 Overview of the WHI Nutritional Biomarkers Study (NBS)

The WHI Nutritional Biomarkers Study (NBS) is a substudy of the WHI. The objective of the NBS is

to collect biological markers of dietary intake (energy, protein, fatty acids, and micronutrients) for use in

regression calibration models that will correct the random and systematic bias of dietary self-report and

better estimate associations of dietary intake with disease outcomes. The calibrated nutrient intake data

will be used for a broad range of nutrient-disease analyses from the WHI. For a copy of the NBS

protocol, refer to NBS Manual, Appendix A – NBS Protocol (ver.2).

NBS will be implemented in a subset of 550 DM women (275 Intervention, 275 Control). DM women

will be mailed an invitation letter prior to their regularly scheduled Annual Visit (AV). Willing

participants will be consented at the first clinic visit (5 hours), complete an FFQ, provide information

about current dietary supplements (Form 45) and physical activity (Form 35), receive a single oral dose

of doubly labeled water (DLW), and provide four spot urines. Women aged 60 years and older will also

provide a blood specimen. Women will return to their CC for a 2-hour visit on about Day 15, at which

time they will return a 24-hour urine specimen (collected at home), be weighed, provide a fasting blood

specimen, and two spot urines. For a subset of 110 women (the reliability subsample), the full protocol

will be repeated in conjunction with a woman’s close-out visit, an average of 6 months later. Women in

this reliability subsample will also complete two 24-hour recalls (24-HR), one within three weeks of

completing the study the first time and the second within about three weeks of the study the second

time. The Nutritional Biomarkers Study implementation begins May 2004 and continues through

March 2005. Figure 1.1 – WHI Nutritional Biomarkers Study Overview provides an overview of NBS.

Figure 1.1

WHI Nutritional Biomarkers Study Overview

CC mails

invitation letters

3-4 weeks

before AV

CC calls participants

within 1 week and uses the

NBS Telephone ScreeningQuestionnaire/Script to

determine eligibility and

interest

Ineligible or Declined

(with Annual Visit)

NBS Visit 1

24 hr. urine

collection

(at home)

Eligible - Schedule NBS Visit 1

Recruitment/Screening

Screening

Results

(Form 74)

Clinic Visits

(2 weeks later)

NBS Visit 2

Reliability Sub-sample

(110 repeat participants, 6 months later)Full Sample (550 participants)

24 HR

recalls

X 2

24 hr. urine

collection(at home)

(with Close-out Visit)

NBS Visit 3

(2 weeks later)

NBS Visit 4

CCC sends

CCsrecruitment

list

NBS Manual


SECTION 2

GUIDELINES

2.0 Overview

This section contains information to help Clinical Centers manage the Nutritional Biomarkers Study.

Guidelines for staffing, training and certification, communication and supplies are provided. This

section may be helpful for staff training and can be used as a checklist to ensure that essential elements

are in place to conduct the study.

2.1 Staffing

Each CC has the following staff positions covered in their initial NBS budget projections: Nutritionist,

Medical Assistant, Research Assistant, and Lab Technician.

Blinding

Ideally staff responsible for data collection (i.e., height/weight, forms review, blood and urine

processing) should be blinded to the DM randomization assignment of NBS participants.

2.1.1 NBS Lead-Ops

Each CC identifies a NBS Lead-ops person to oversee NBS operations and be the NBS point person.

This person may be the CC Principal Investigator, the investigator with local oversight for the NBS, or a

separate person. The NBS Lead-ops person needs to:

Be in the clinic and involved in the NBS visits so that they can supervise what is happening and

respond to questions that staff may ask.

Train other non-lead NBS staff.

Ensure that the NBS protocol is followed, data entry is timely, supplies are available and any

procedural or data entry problems are reported to the CCC.

Oversee management of the following NBS operations:

o Recruitment

o Integration of NBS visit tasks with AV and WHI Close-out tasks

o NBS visits and tasks

o CC-NBS budget

2.2 Training and Certification

The purpose of training within NBS is to teach staff the NBS protocol and procedures for

implementation at the Clinical Center (CC). To facilitate the implementation of NBS, the CCC uses a

training model similar to the one used in WHI. One NBS Lead-ops person per CC receives training for

all NBS-specific tasks and is then responsible for training other staff for NBS tasks. The CCC provides

training conference calls, the NBS Manual, and NBS Staff Training and WHI Certification Guidelines

(refer to NBS Manual, Appendix E – NBS Training Materials).

2.2.1 NBS Lead-Ops Person

The NBS Lead-ops person attends each of the four NBS training calls and reads the NBS Manual.

If the Lead NBS-ops person cannot attend a NBS training call, ‘essence’ transcriptions of the

calls are available. To be trained and certified, the Lead NBS-ops person reviews the transcript

of the training call(s) and reads the NBS Manual.

NBS Manual


2.2.2 Non-Lead NBS Staff

Non-lead NBS staff read relevant sections of the NBS Manual and discuss relevant training call topics

with the NBS Lead-ops person. To help guide training for non-lead NBS staff, copies of the ‘essence’

transcriptions of the four NBS training calls are available in the NBS Manual, Appendix E – NBS

Training Materials.

For WHI tasks that are part of the NBS (i.e., physical measures, FFQ review, blood drawing and

processing, forms review, and data entry), the CCC asks that CCs use WHI-certified staff. For

guidelines on which tasks need WHI certification, refer to WHI Certification Guidelines in the NBS

Manual, Appendix E – NBS Training Materials.

For training on NBS-specific urine collection and processing procedures, the NBS Lead-ops person

attends the NBS training calls and reads the NBS Manual. The Lead-ops person trains the designated

CC staff. CCs must also follow the pertinent safety precautions found in the NBS Manual, Section

7.1.2– Safety Procedures – Precautions for Handling Urine, as well as their local regulations for bio-

specimen handling (blood and urine).

2.2.3 Training Resources

2.2.3.1 NBS Training Conference Calls

The CCC conducts four NBS training conference calls at the time of NBS start-up.

Call #1: provides an introduction and overview of NBS (purpose of study, recruitment, visit

tasks and housekeeping issues for start-up).

Call #2: focuses on the doubly labeled water (DLW) protocol.

Call #3: focuses on specimen collection and processing of spot and 24-hour urine and blood

biomarkers, as well as forms and data entry.

Call #4: provides an opportunity to review and discuss the overall NBS process.

The NBS Manual, Appendix E – NBS Training Materials includes the agenda and ‘essence’ transcription

for each training conference call.

2.2.3.2 NBS Procedure Manual (referred to as the NBS Manual)

The NBS Manual describes the study design (as outlined in the study protocol), procedures, and data

management. The primary function of the manual is to provide common training and reference

materials across all participating NBS clinics as a way of assuring the quality of the study.

2.3 Communication

2.3.1 Email Communication

The following types of email communication are sent to the CC PI, all NBS CC investigators, and

individuals identified to receive NBS mailings (i.e., NBS Lead-ops person).

NBS Updates: A weekly email to maintain systemic communication and keep NBS clinics

informed.

NBS Alerts: Priority email sent when there is immediate information that is important to quickly

relay to all NBS clinics.

CCs send NBS-related questions to Helen Penor ([email protected]) for triaging to the appropriate CCC

staff for a response.

mailto:[email protected]

NBS Manual

WHI NUTRITIONAL BIOMARKERS STUDY (NBS) VER 2: JULY 2004

2.3.2 WHI Public Folders

The Outlook Public Folders contain a Nutri Biomarker Study folder for NBS-specific materials and

communications. This folder provides resources for the implementation of the NBS. The Nutri

Biomarker Study folder includes items such as:

NBS WHILMA Upgrade notes.

NBS Updates – copies of all NBS weekly updates published to date.

NBS Alerts – copies of all NBS Alerts published to date.

NBS materials submitted to the FHCRC IRB – copies of materials submitted and approved by the

FHCRC IRB.

NBS Timeline.

NBS Study Materials – electronic copies of all data collection forms, worksheets and participant

materials unique to NBS.

NBS Q&A about Study Procedures

2.4 Supplies

This section provides information about supplies for the WHI Nutritional Biomarkers Study (NBS).

2.4.1 Supplies Obtained by Each CC

Each CC obtains the supplies shown below. The CCs will receive reasonable reimbursement for items

necessary to complete the study.

Urine Collection: 1

Gel ice packs

Safety pins

Miscellaneous: 1

Participant meal replacement beverage

Participant lunch

Participant snack

Straws

Ziplock baggies

Print Materials

CCs print NBS forms and materials locally from masters provided by CCC. Electronic copies of the masters

are posted in the Outlook Public Folders/Nutri Biomarkers Study/NBS Study Materials. Hard copies of the

masters are also available in the NBS Manual (Section 9 – NBS Forms and Worksheets, Appendix C – NBS

Participant Materials and Appendix D – NBS Staff Materials). The NBS forms and materials that CCs print

locally are outlined below.

Letter of Invitation to Participants

Telephone Screening Questionnaire/Script

Frequently Asked Questions (for staff)

Study at a Glance

NBS Consent

Instructions for 24-hour Urine Collection

NBS Visit Forms (Forms 74 - 78)

NBS Visit 1 Eligibility Checklist Worksheet

NBS Visit 2 Participant Update Worksheet

NBS Visit 2 (or Visit 4) PABA Worksheet for Staff



Consent for Future Contact and Continued Participation in this Study

NBS Manual


Other Supplies (WHI supplies used for NBS):

Specimen collection and processing supplies:1

For example: Royal blue-stoppered serum tubes, Lavender-stoppered plasma tubes, 2.0 mL

cryovials, gloves, needles, disposable pipette tips.

Office supplies (e.g., envelopes, mailing labels, member ID labels, postage)

1

For information about recommended vendor/part number and quantities to purchase, refer to NBS Manual,

Section 2.4.4 – Guidelines for Ordering Supplies.

2.4.2 Supplies Provided by the CCC

The CCC provides each CC with the supplies outlined below.

Forms & Folders & Labels

WHI Folders

NBS label set

WHI Form 35

WHI Form 45 (Back-up)

WHI Form 60 2

Urine Collection:

Urine collection hats

24-hour urine collection containers (3 liter)

Plastic funnels

Sturdy plastic bags with a handle

For participants who receive PABA:

PABA (B-vitamin) tablets, 3 per participant

Boric acid powder (preservative for the 24-hour urine collection)

Stickers for 24-hour urine collection containers

Urine Processing:

5 mL Corning cryovial tubes with rubber O-ring seal

250 mL graduated cylinders

10 mL tubes (for centrifuging 24-hour urine collection sample)

Disposable pipettes (for urine)

Urine Storage and Shipping:

Freezer boxes with dividers for the 5 mL Corning cryovial tubes

2 Note: The CCC will provide Form 60 (Ver. 1.5) which can only be marked for Visit Type AV1 – AV9.

CCs will use Form 60 (Ver. 1.6) from their local supply if an NBS participant is at AV10 or AV11 and part of

the FFQ subsample. FFQ Ver. 1.5 and Ver. 1.6 are identical except that Ver. 1.6 can be marked for Visit

Type AV10 or AV11.

2.4.3 Supplies Provided by Dale Schoeller (University of Wisconsin)

Dale Schoeller at the University of Wisconsin provides each CC with the supplies outlined below.

Doubly Labeled Water (DLW) Protocol:

DLW (in pre-measured bottles)

Pre-weighed tissues (for use in case of DLW spillage)

NBS Manual


2.4.4 Guidelines for Ordering Supplies

The list below includes guidelines for ordering miscellaneous supplies and supplies for blood and urine

collection, processing, and shipment. CCs may choose to use a different vendor than the one

recommended for supplies obtained locally. Please note that blood collection supplies may be ordered

through McKesson BioServices, as indicated.

Miscellaneous Supplies Recommended Vendor/Part Number:

NBS-specific:

Participant meal replacement beverage (8 ounce) for

1 hour after DLW dose at Visit 1

1 per NBS participant

Local medical supply or store

Suggested brand: Boost (based on

informal taste testing at the CCC)

Participant lunch at the end of Visit 1


CC option

Participant snack at the end of Visit 2


CC option

Straws (for drinking DLW)



(a small size that can be pushed all the

way inside the DLW dose bottle)

Ziplock baggies (for use with pre-weighed tissues in

case of DLW spillage)

1 box (of ~50) per CC

CC to purchase at local store

Blood and Urine Collection Supplies

NBS-specific:

Urine collection hats (Specipans)

3 per NBS participant – 1 to use at each of two clinic

visits, 1 to send home with participant for 24-hour

urine collection

CCC will order and provide

24-hour urine collection containers

2 (3 liter) per NBS participant


Plastic funnels for 24-hour urine collection



Plastic carry bag for 24-hour urine collection kit



Safety pins (large size) for participants to pin to their

undergarments as a reminder for 24-hour urine

collection

1 per NBS participant (1 box of ~100 per CC)

Local store

Gel ice packs for participants to transport their 24-hour

urine collections to the Clinical Center

20 per CC (~6 ounce; 4” x 6”)


For participants who receive PABA

PABA (B-vitamin) tablets, 3 per participant


Boric acid powder, one 500 gram jar CCC will order and provide

Stickers for 24-hour urine collection containers CCC will order and provide

NBS Manual


Blood and Urine Collection Supplies Recommended Vendor/Part Number:

Same as WHI (use from WHI stock and order as usual to

replenish supplies):

Royal blue-stoppered serum tubes for trace elements

and carotenoids, no additive, silicone coated, 7 mL

(Note: 7 mL will be printed on label in red ink; be

sure you use the tubes with 7 mL in red and not

7 mL in green)


McKesson or BD 367737 (Baxter B2951-

107, Vacutainer tubes with hemoguard

closure) or BD 6526 (Baxter B3007-54)

Lavender-stoppered plasma 10 mL tube, with

powdered Sodium (Na2 EDTA)

1 per NBS participant under the age of 60 years

2 per NBS participant 60 years of age and older

McKesson or Monoject 310745 Terumo

Venoject T200SQ (Baxter B3042-54 or

Laboratory Supply)

Needles, 21 gauge, 1 to 1½” multiple sample

Vacutainer

Local medical supply

23-gauge butterfly with 12” tubing with multiple

sample Luer adapter


Syringes

Monoject hypodermic needle (21-23 gauge, 1 ½”)

Blood draw workstation Recommended Baxter S9267-1

Vacutainer holder Local medical supply

Test tube racks Local medical supply

Alcohol swabs or cotton balls, alcohol, and alcohol

dispenser or gauze swabs


Bandages (“Band-Aids”) or surgical tape Local medical supply

Biohazard container for needles (“sharps” container) Local medical supply

Biohazard container for waste Local medical supply

Wash bottle Local medical supply

Lab coat Local medical supply

Disposable latex gloves Local medical supply

Chlorine bleach Local store

Anti-bacterial hand soap Local store

Aluminum foil or yellow plastic sleeves to protect the

blood drawn in all of the blood collection tubes from

light (NBS Visit 2)

Local store

NBS Manual


Blood and Urine Processing Supplies Recommended Vendor/Part Number:

NBS-specific:

5 mL Corning cryovial tubes with rubber O-ring for

processing spot urine samples


12 additional for each participant who has two

additional aliquots taken at each time point for QC (for

QC blind duplicates, aliquot one extra vial for each

collection for each of 30 participants NBS-wide)


250 mL graduated cylinder to measure 24-hour urine

samples

2 per CC


10 mL tube for centrifuging 24-hour urine collection

samples

1 per participant per CC plus 10 extra


Disposable transfer pipettes (for transferring urine from

DLW spot urine collection to 5 mL cryovial, and for

transferring the 10 mL centrifuged 24-hour urine sample

into the three 2 mL cryovials)

7 per participant



replenish supplies):

2 mL cryovials for serum, plasma and 24-hour urine

13 total:

Visit 1

3 for Lavender tube 3-hour post-DLW blood draw

(for women 60 years and older)

Visit 2

4 for Royal Blue fasting blood

3 for Lavender fasting blood

3 for 24-hour urine

Fluid-resistant lab coat Local medical supply

Refrigerated centrifuge with swinging buckets

(able to reach relative centrifugal force of 1,300 xg)


Factory certified low temperature thermometer, -90ºC

to +20ºC or thermistor

Large and small test tube racks for holding Vacutainer

tubes and cryovials


Disposable latex gloves Local medical supply

Chlorine bleach Local medical supply

Goggles or glasses or mask with face shield or barrier

shield behind which to process blood samples


Tape Local store

1 mL adjustable automatic pipettor Local medical supply

NBS Manual


Blood and Urine Processing Supplies (continued) Recommended Vendor/Part Number:

Disposable pipette tips (for blood processing) Local medical supply

10 x 75 mm or larger test tube or 15 mL conical centrifuge

tube to recentrifuge samples

Baxter C3902-4

Blood and Urine Storage and Shipment

NBS-specific:

Freezer boxes for 5 mL Corning cryovial tubes CCC will order and provide


replenish supplies. Exceptions: freezer, low temperature

thermometer, and scale which are pre-existing WHI equipment)

Freezer at 70°C or colder, with CO2 back-up system

and temperature recorder

Pre-existing WHI equipment

Factory certified low temperature thermometer, -90C

to +20C or thermistor

Pre-existing WHI equipment

Newspaper (for packing)

Freezer alarm for each -70C freezer Rees Scientific/Informer 2400

Telephone: (800) 327-3141

Waterproof packing tape (strapping tape)

Dry ice nuggets

Freezer gloves/vinyl or latex rubber gloves

Return address label (printed at CC)

Indelible ink pen

Shipping tape

Scale for weighing shipment Pre-existing WHI equipment

NBS Manual Page


10

SECTION 3

RECRUITMENT

3.0 Overview

This section contains information to help clinical centers manage NBS recruitment. The section

describes the NBS study sample, recruitment goals, recruitment plan, recruitment list, invitation letter,

WHILMA resources for recruitment, and procedures for expanded NBS recruitment (beginning July

2004).

3.1 NBS Study Sample

NBS will be implemented in a subset of 550 DM women (275 Intervention, 275 Control) with age and

race/ethnicity strata that represent the composition of the DM cohort.

3.2 Recruitment Goals

The recruitment goal for each CC is approximately 50 participants, except the two minority CC sites,

which are expected to recruit at least 25 minority participants each.

Each CC will strive to recruit 25 DM-I participants and 25 DM-C participants (minority sites about 13

DM-I and 13 DM-C participants). CCs do not have goals for the various age and ethnicity strata. The

CCC will close strata when the goal for a specific stratum has been reached NBS-wide (e.g., white

women age 55-59). Minority cells will remain open for recruitment after the goal has been met to allow

as many minorities as possible to participate. Non-minority cells will close after the goal has been met.

3.3 Recruitment Plan

Two NBS recruitment plans are available as of July 2004:

NBS Visit linked with AV

Expanded NBS recruitment

NBS Visit Linked with AV:

The plan for NBS recruitment is to invite and schedule participants for NBS in conjunction with their

WHI Annual Visit (AV). Specifically, the plan includes approaching only those participants who are

due for their AV during the NBS window, but have not yet completed the AV. This recruitment plan is

designed to:

Minimize participant burden.

Avoid subjectively selecting participants (to avoid selection bias).

Ensure that the age and race/ethnic composition of the NBS sample is reasonably representative

of the DM cohort.

Ensure that women known to have exclusion criteria (e.g., diabetes) do not receive an invitation

letter.

It is important for all CCs to begin recruitment by using the NBS recruitment plan (i.e., approach only

participants who are due for their AV during the NBS window, but have not yet completed their AV).

The CCC will monitor recruitment rates and will re-evaluate CC recruitment in early July 2004 to see if

there is a need to expand the recruitment pool.

NBS Expanded Recruitment:

For information about expanded NBS recruitment beginning July 2004, refer to the NBS Manual,

Section 3.7 – Expanded NBS Recruitment (Beginning July 2004).

NBS Manual Page


11

3.4 Recruitment List

The CCC provides each CC with a list of eligible participants by way of the NBS Recruitment Tracking

report (NBS001). The report includes DM participants (Intervention and Control) likely to have their

AV during the NBS window (May-September 2004) and who meet the eligibility criteria outlined in the

NBS protocol. The list includes women who meet the following criteria:

Have an AV target date between February-September 2004.

Have not completed an AV for 2004.

Have a FFQ at baseline, Year 1, and at least one additional time point.

Have full-follow-up status.

Do not take insulin or oral hypoglycemic agents to manage diabetes.

Are not part of the WHI blood draw cohort.

CCs use the NBS Recruitment Tracking report (NBS001) to invite and schedule women for NBS in

conjunction with their AV. For further details about the recruitment list and the NBS Recruitment

Tracking report, refer to NBS Manual, Section 3.6 - WHILMA Resources for NBS Recruitment Tracking

and the Nutritional Biomarkers Study-Recruitment Upgrade Notes located in the Outlook Public

Folders/All Public Folders/Nutri Biomarkers Study (dated 5/12/04).

For information about the supplemental list for expanded NBS recruitment, refer to the NBS Manual,

Section 3.7.3 – Supplemental Recruitment List for Expanded NBS Recruitment.

3.5 Invitation Letter

After receiving the participant recruitment list from the CCC, CCs begin to mail the NBS invitation

letter to participants in weekly batches (i.e., ideally about 3 weeks before the AV, but no less than 2

weeks). A copy of the NBS Letter of Invitation to Participants is available the NBS Manual, Appendix

C – NBS Participant Materials.

It is important to mail the NBS invitation letter separate from other AV mailings. This provides an

opportunity to:

Ensure that the invitation letter is not lost in the WHI AV materials.

Send the invitation letter in a timely manner (i.e., about 3 weeks before the AV).

It is important to mail the invitation letter in small batches (i.e., do not to mail to the entire NBS

recruitment list all at once). If CCs mail too far in advance, the participant’s strata status may change

before a CC can contact the participant. Use the ‘AV Target Date Between’ parameter on the NBS

Recruitment Tracking report (NBS001) to identify eligible participants due for an upcoming AV. NBS

staff can choose an AV target date range to identify participants between XX and XX dates. This will

allow CCs to mail smaller weekly batches of invitation letters to eligible participants.

To avoid any potential of selection bias (subjectively selecting participants), CCs should mail an

invitation letter to only the participants appearing on their NBS Recruitment Tracking report (NBS001)

(i.e., don’t add names) and to invite all participants appearing on their NBS Recruitment Tracking report

(NBS001)unless:

They have completed their AV visit.

Their strata closes before the CC approaches them.

They are known apriori to be ineligible (e.g., live greater than 200 miles distant from the CC).

Refer to NBS Manual, Section 4.1.4 – Complete and Data Enter NBS Screening Result (Form 74).

NBS Manual Page


12

To track the NBS mailings and screening calls, each CC sets up a system to track the following pieces

of recruitment information:

Who has been sent the NBS invitation letter.

Date letter sent.

Date to call participant (one week after invitation letter sent).

Number of phone attempts (refer to NBS Manual, Section 4.1.1 - Telephone Contact).

For information about a sample Call Attempts Log, refer to Nutritional Biomarkers Study-

Recruitment Upgrade Notes located in the Outlook Public Folders/All Public Folders/Nutri

Biomarkers Study (dated 5/12/04).

For information about approaching participants for expanded NBS recruitment, refer to the NBS

Manual, Section 3.7.4 – Approaching Participants for Expanded Recruitment.

3.6 WHILMA Resources for NBS Recruitment Tracking

This section describes the resources for NBS recruitment tracking. This includes the NBS Recruitment

Tracking report (NBS001) and the NBS Custom Data Extract (CDE for NBS001). Supporting

information can be found in the Nutritional Biomarkers Study-Recruitment Upgrade Notes located in

the Outlook Public Folders/All Public Folders/Nutri Biomarkers Study (dated 5/12/04).

For information about the NBS Supplemental Recruitment Tracking report (NBS002), refer to NBS

Manual, Section 3.7.3 – Supplemental Recruitment List for Expanded Recruitment.

3.6.1 NBS Recruitment Tracking Report

The NBS Recruitment Tracking report (NBS001) provides NBS staff with a complete system for

tracking NBS recruitment. For information about the report and the parameters that allow the data to be

sorted and filtered, refer to Nutritional Biomarkers Study-Recruitment Upgrade Notes located in the

Outlook Public Folders/All Public Folders/Nutri Biomarkers Study (dated 5/12/04).

Helpful Hints:

NBS staff can run the NBS Recruitment Tracking report (NBS001) in two different formats: a

‘List’ format which provides a list of several participants per page or a ‘Call Log’ format which

lists one participant per page and includes a blank Call Attempts Log. NBS staff may find the

Call Attempts Log helpful for tracking their screening call attempts to participants.

To ensure that women are invited and scheduled in conjunction with their AV, use the ‘Maybe

Had AV’ column on the NBS Recruitment Tracking report (NBS001). A ‘yes’ in this column,

identifies participants who have completed Form 80 or Form 33 for the targeted AV. If there is a

‘yes’, check to see if the woman has already completed her AV. If she has completed her AV, do

not mail an invitation letter.

CCs should monitor the ‘Strata Status’ column on the NBS Recruitment Tracking report

(NBS001) as they prepare their mailings to avoid mailing to participants who should not be

contacted because their strata has closed.

The NBS Recruitment Tracking report (NBS001) can be run with a participant’s randomization

assignment (i.e., Intervention or Control) either showing or hidden. The ability to ‘hide’ DM

randomization assignment provides flexibility and the option to maintain DM blinding for NBS

staff not involved in NBS recruitment.

NBS Manual Page


12a

3.6.2 NBS Custom Data Extract

The NBS Custom Data Extract (for NBS001) contains the same columns as the NBS Recruitment

Tracking report (NBS001), plus some additional information (e.g., a column that flags participants who

agree to repeat the NBS protocol in 6 months).

CCs can use the NBS CDE to generate NBS mailing labels and participant member ID labels for the

front page of NBS forms. CCs familiar with the CDE system may also use the NBS CDE for tracking

NBS recruitment and NBS visit status, if they choose.

3.7 Expanded NBS Recruitment (Beginning July 2004)

This section describes procedures for expanded NBS recruitment beginning July 2004. CCs choosing to

expand recruitment need to investigate approvals that their local IRB may require.

3.7.1 Expanded Recruitment Plan

Beginning July 2004, the plan for NBS recruitment expands to allow recruitment of non-AV NBS

participants.

This expanded recruitment plan is designed to:

Support CC efforts to meet NBS recruitment goals.

Continue to meet the criteria outlined in Section 3.3 - Recruitment Plan:

Minimize participant burden.

Avoid subjectively selecting participants (to avoid selection bias).

Ensure that the age and race/ethnic composition of the NBS sample is reasonably

representative of the DM cohort.

Ensure that women known to have exclusion criteria (e.g., diabetes) do not receive an

invitation letter.

3.7.2 NBS Study Materials for Expanded Recruitment

The NBS protocol and study materials have been modified to allow expanded recruitment. The FHCRC

IRB-approved revised materials can be found in the NBS Manual as follows:

See NBS Manual, Appendix A – Protocol for the NBS Protocol, Version 3, July 2004

See NBS Manual, Appendix C – NBS Participant Materials for NBS Letter of Invitation,

Version 41

See NBS Manual, Appendix D – NBS Staff Materials for the NBS Telephone Screening

Questionnaire/Script, Version 2, July 2004

See NBS Manual, Appendix B - NBS Consents for NBS Consent, Version 4, July 20041

See NBS Manual, Appendix C - NBS Participant Materials for Study At A Glance, Version 4,

July 20041

See NBS Manual, Appendix C – NBS Participant Materials for Instructions for 24-hour

Urine Collection, Version 3, July 2004

1Clinical centers conducting indirect calorimetry procedures at NBS Visit 2 (Chicago-Northwestern,

Madison, and Pittsburgh) will use a different letter of invitation, consent, and Study at a Glance. For

copies of the materials to be used only by CCs conducting indirect calorimetry procedures, refer to

NBS Manual, Section 10 – Indirect Calorimetry, Appendix for Section 10.

Clinical centers choosing to expand their NBS recruitment pool will use the revised materials and will

have local IRB approval as necessary.

NBS Manual Page


12b

3.7.3 Supplemental Recruitment List for Expanded Recruitment

In July 2004, the CCC provides each CC with a supplemental list of participants eligible for NBS by

way of the NBS Supplemental Recruitment Tracking report (NBS002). The NBS Supplemental

Recruitment Tracking report (NBS002) includes DM participants (Intervention and Control) who meet

the following criteria:

Have a FFQ at baseline, Year 1, and at least one additional time point.

Have full-follow-up status.

Do not take insulin or oral hypoglycemic agents to manage diabetes.

Are not included on NBS Recruitment Tracking report (NBS001).

For information about accessing the NBS Supplemental Recruitment Tracking report (NBS002) in

WHILMA, refer to the WHILMA Upgrade Notes for NBS Supplemental Recruitment Tracking Report

located in the Outlook Public Folders/All Public Folders/Nutri Biomarkers Study (dated 7/26/04).

3.7.4 Approaching Participants for Expanded Recruitment

If a clinical center chooses to expand NBS recruitment, the NBS recruitment pool may be expanded in

two ways:

Invite participants from the NBS Recruitment Tracking report (NBS001) whose Annual Visits

(AVs) have already occurred.

Invite participants from the NBS Supplemental Recruitment Tracking report (NBS002).

Invite only participants who are listed on the two NBS recruitment tracking reports (described

immediately above). Participants whose names are not on the recruitment tracking reports cannot be

invited to participate in NBS. The recruitment tracking reports pre-exclude WHI DM participants who

do not meet the NBS pre-telephone screening eligibility criteria.

To avoid any potential of selection bias (subjectively selecting participants), identify participants to

invite using the procedures outlined below.

When inviting participants from the NBS Recruitment Tracking report (NBS001):

Use the ‘AV Target Date Between’ parameter to choose an AV target date range to identify

participants between X and XX dates. Invite all participants in the selected date range.

Exception: do not invite participants whose strata have closed.

If you are still recruiting for the NBS reliability subsample (NBS Visits 3 and 4), choose only

an AV target date range between July and September 2004. These participants will have

Close-out visit target dates between January and March 2005. This will ensure that there is

adequate time between NBS Visits 1 and 2 and the NBS reliability subsample visits.

If you have finished recruiting participants for the reliability subsample, but still need

participants for NBS Visits 1 and 2, choose any date range to identify participants.

Choose a date range that allows mailing in small weekly batches; i.e., a weekly mailing of

invitation letters should include only the number of participants that can reasonably be contacted

within one week of the mailing.

NBS Manual Page


12c

When inviting participants from the NBS Supplemental Recruitment Tracking report (NBS002):

Use the ‘Close-out Target Date Between’ parameter to choose a Close-out target date range to

identify participants between X and XX dates. Invite all participants in the selected date range.

Exception: do not invite participants whose strata have closed.

If you are still recruiting for the NBS reliability subsample (NBS Visits 3 and 4), choose only

a Close-out visit target date range between January and March 2005. This will ensure that

there is adequate time between NBS Visits 1 and 2 and the NBS reliability subsample visits.

If you have finished recruiting participants for the reliability subsample, but still need

participants for NBS Visits 1 and 2, choose any date range to identify participants.

Choose a date range that allows mailing in small weekly batches; i.e., a weekly mailing of

invitation letters should include only the number of participants that can reasonably be contacted

within one week of the mailing.

NBS Manual Page 13


SECTION 4

SCREENING

4.0 Overview of Screening Process

The NBS screening process determines the eligibility and interest of potential DM participants. Below

is a summary overview of the screening process:

Contact each participant by phone within one week of mailing the invitation letter.

Use the Telephone Screening Questionnaire/Script to determine the participant’s eligibility and

interest.

Schedule a NBS Visit 1 for eligible and interested participants.

4.1 Screening Activities

4.1.1 Telephone Contact

CCs telephone all participants who are mailed an invitation letter within one week of the NBS mailing.

A NBS interviewer uses the Call Attempts Log in the NBS Recruitment Tracking report (NBS001), NBS

Supplemental Recruitment Tracking report (NBS002), or a similar CC-designed sheet to record the dates

when a participant is contacted by phone.

The CCC strongly recommends that a CC’s Call Attempts Log contain the following information:

date, time of day, day of the week, and a place for interviewer’s comments.

For information about a sample Call Attempts Log, refer to Nutritional Biomarkers Study-

Recruitment Upgrade Notes located in the Outlook Public Folders/All Public Folders/Nutri

Biomarkers Study (dated 5/12/04).

If the NBS interviewer is unable to reach the participant by telephone, after at least five attempts (at

different times of the day and days of the week) the participant is marked ‘Ineligible’ on Form 74 –

Screening Result (refer to NBS Manual, Section 4.1.4 – Complete and Data Enter NBS Screening Result

(Form 74).

4.1.2 Determine Eligibility and Interest

During the screening phone call, the NBS interviewer uses the Telephone Screening

Questionnaire/Script to explain the NBS study and determine the participant’s eligibility and interest. If

needed, the interviewer may refer to Frequently Asked Questions document when answering

participants’ questions about the doubly labeled water (DLW) protocol. Copies of the Telephone

Screening Questionnaire/Script and the Frequently Asked Questions are located in the NBS Manual,

Appendix D – NBS Staff Materials.

4.1.3 Schedule NBS Visit 1

If a participant is eligible and interested, the NBS interviewer schedules the NBS Visit 1 to coincide

with the participant’s WHI AV. Whenever possible, every effort should be made to avoid scheduling

Dietary Change (Intervention) and Comparison (Control) women on the same day to avoid

‘contamination’ of the WHI DM Comparison group.

NBS Manual Page 14


4.1.4 Complete and Data Enter NBS Screening Result (Form 74)

The NBS interviewer completes a Form 74 for all participants mailed the NBS invitation letter.

The NBS interviewer records the participant’s ‘Final Screening Disposition’ on Form 74 – NBS

Screening Result (Qx. 5 - Final Screening Disposition). The ‘final disposition’ represents the

participant’s status at the end of the recruitment/screening phone call:

‘Scheduled’: Participant was eligible and interested in the NBS study and is scheduled for NBS

Visit 1.

‘Ineligible’: Participant was not eligible to participate in the NBS study.

‘Declined’: Participant was not interested in participating in the NBS study.

There are a few circumstances where the CC has the option to complete a Form 74 for a participant

without mailing the NBS invitation letter and conducting the NBS Telephone Screening

Questionnaire/Script.

Circumstance #1:

The CC knows with certainty that the participant will be ineligible per the eligibility questions on

the NBS Telephone Screening Questionnaire/Script (e.g., the participant has moved away from the

CC and would need to travel more than 200 miles for the NBS visit). In this case, the CC has the

option to complete the Form 74 for the participant without mailing the invitation letter and

conducting the NBS Telephone Screening Questionnaire/Script (mark Form 74, Qx. 5 – Final

Disposition as ‘2 – Ineligible’).

Circumstance #2:

For participants who have significant cognitive challenges (e.g., a diagnosis of dementia) or whose

WHI participation may be jeopardized by receiving an NBS invitation letter, CCs have the option

to complete the Form 74 for the participant without mailing the invitation letter and conducting

the NBS Telephone Screening Questionnaire/Script (mark Form 74, Qx. 5 – Final Eligibility

Disposition as ‘2 – Ineligible’). Caution: please exercise this discretionary removal of potentially

eligible participants only when absolutely necessary. It is important that we avoid potential bias in

our sample by not ‘cherry picking’ and by giving each participant the benefit of the doubt and the

opportunity to self-select ‘declined’ participation.

Key-enter Form 74 only after the final result of the telephone screening call is known. For example, if a

participant is unable to schedule her NBS Visit 1 appointment until she checks her family schedule, then

Form 74, Qx. 5 – Final Eligibility Disposition would not be marked until the participant has been

scheduled for NBS Visit 1.

Key-enter the Form 74 - NBS Screening Result within 1 working day of completing the form. The CCC

will use information from Form 74 to monitor the DM strata goals for age and race/ethnicity.

File the completed and data entered Form 74 in the participant’s chart (or special NBS notebook).

NBS Manual Page 15


SECTION 5

NBS VISITS

5.0 Overview of NBS Visits

There are two NBS Visits (NBS Visit 1 and NBS Visit 2): Figures 5.1 and 5.3 – provide an overview of

the specific tasks for NBS Visit 1 and NBS Visit 2. Sections 5.1 - NBS Visit 1 (Day 1) and 5.2 – NBS

Visit 2 (Day 15) describe the activities of the two visits in detail and follow the flow depicted in Figure

5.1 and Figure 5.3.

In general, all visits contain similar activities in terms of preparation for the visit, greeting the

participant, performing various visit activities, reviewing forms, providing the participant with

appropriate materials before she leaves, scheduling the next visit, and data entry. Because the visits

contain many different activities and the participant will most likely be seen by several different CC

staff, it is critical to designate a NBS staff person to oversee the NBS visits (including pre- and post-

visit activities).

5.1 NBS Visit 1 (Day 1)

5.1.1 Overview of Activities

Figure 5.1 – Overview of NBS Visit 1 (Day 1) provides an overview of NBS Visit 1.

NBS Manual Page 16


Figure 5.1

Overview of NBS Visit 1 (Day 1)

Participant Arrival

Greet participant

Review the NBS study components and visit activities

Determine eligibility (NBS Visit 1 Eligibility

Checklist Worksheet)

Review NBS Consent

NBS Consent signed NBS Consent not signed

Eligible Ineligible

Give participant a copy of signed NBS Consent

Give participant Study At A Glance handout

Obtain height and weight

Time 0 (fasting) spot urine: obtain and process

Give DLW (doubly labeled water) to drink

Provide forms to participant to complete: Form 35 (Personal Habits

Update) and Form 60 (FFQ)

Complete Task 45 – Current Supplements or

use Form 45 (Back-up)

1 hour post DLW:

Provide meal replacement beverage

Participant continues working on Forms 35 and 60

2 hours post DLW:

Obtain and process spot urine

3 hours post DLW:


Obtain and process blood for women > 60 years of age

Collect, review and scan Form 35 (Personal Habits Update)

and Form 60 (FFQ) before participant leaves the clinic

4 hours post DLW:


Instruct participant on 24-hour urine collection and

provide materials for collection

Schedule participant for NBS Visit 2 (15 days later)

Provide complementary meal

Complete blood and urine processing and storage

Key enter Form 75 - NBS Visit 1

NBS Manual Page 17


5.1.2 Integration of NBS Visit 1 and Annual Visit

NBS Visit 1 occurs in conjunction with the WHI Annual Visit (AV). The NBS Visit 1 is integrated with

the AV tasks so that all activities are completed as required and participant burden is minimized. Figure

5.2. – Integration of NBS Visit 1 and Annual Visit Tasks provides the estimated amount of time for each

NBS task and shows how NBS Visit 1 and AV tasks could be integrated.

It is important to BEGIN the participant’s NBS Visit 1/AV with the NBS tasks and to MAINTAIN the

sequence of NBS activities throughout the visit. This is important because:

The length of time a participant is in the clinic depends on when the NBS activities begin.

The sooner the NBS activities begin, the sooner the participant completes her clinic visit.

Having a NBS staff person be the first person to see the participant when she arrives, allows the

‘visit eligibility’ questions to be asked and the participant to begin NBS visit activities, if she

remains eligible.

The sequence of the NBS activities related to the doubly labeled water dosing and urine/blood

collections is critical to the protocol and should not be changed.

The NBS protocol and AV tasks will take approximately 5 hours for participants to complete. DM-only

participants may have some free time between tasks. When scheduling participants, encourage them to

bring something to keep busy (e.g., crossword puzzles, a book, knitting, etc.). Women who are in Year

9 and/or other protocols (e.g., bone density protocol, ancillary studies) may need to stay longer than 5

hours.

While the sequence of the NBS activities may not be changed, the sequence of the AV tasks may be

modified to meet CC needs. It is critical to keep track of the time for NBS tasks. Consider using a stop

watch or other reminder to document the first fasting urine sample, doubly labeled water dosing, and

tasks scheduled at specific intervals thereafter (i.e., meal replacement beverage, spot urine collections,

and blood draw for women > 60 years of age).

It is also important to have a designated NBS staff person oversee the NBS/AV activities – pre and post

visit. Responsibilities could include:

Checking the Tasks Required at Follow-up Visit report (WHIP 0144) to identify NBS participants

scheduled to complete Forms 35, 45, or 60 as part of their AV visit.

Verifying that participants scheduled to complete Forms 35, 45, or 60 as part of their AV have

completed the form(s). If a participant scheduled to complete Forms 35, 45, or 60 as part of their

AV forgets to bring the completed form to her AV, ask her to complete the form before she

leaves the CC.

Ensuring that all WHI forms required by the NBS protocol (i.e., Forms 35, 45, or 60) are

collected, reviewed, and scanned before the participant leaves the clinic.

NBS Manual Page 18


Figure 5.2 – Integration of NBS Visit 1 and Annual Visit Tasks

* The sequence of the tasks in the starred columns may be modified to best meet your clinic’s needs.

45 min

Estimated

Times

NBS Visit Tasks DM Annual

Visit Tasks*

CaD & HT Annual

Visit Tasks*

Year 9 Annual

Visit Tasks*

5 min Reception Bone Density sites only -

Collect first morning urine

sample

5 min Complete NBS Visit 1 Eligibility

Checklist Worksheet

20 min Complete NBS Consent

5 min Obtain- height and weight

10 min Time 0 - Fasting spot urine

collection and processing

~ 60 min

5 min Time 0 - Doubly Labeled Water

Dosing –participant sits still for 15

minutes - completes forms

5 min Obtain general medical

releases

20 min Current Supplements

(Task 45)

10 min Personal Habits Update

(Form 35)

20 min Current Medications

(Form 44)

5 min Time 1 - Meal replacement

beverage consumed: 1 hour after

DLW

~ 60 min

30 min FFQ (Form 60) – participant

completes

20 min Breast exam – optional

10 min Time 2 – Spot urine collection and

processing: 2 hours after DLW

NBS Manual Page 19


Figure 5.2 – Integration of NBS Visit 1 and Annual Visit Tasks (continued)


~ 60 min

Estimated

Times


Visit Tasks*

CaD & HT Annual

Visit Tasks*

Year 9 Annual

Visit Tasks*

15min Functional status - sub

sample grip strength, chair

stand, and timed walk (25%

over 65)

5 min HT - Complete HT

management and safety

interview (Form 10)

10 min HT - Cognitive assessment

for ppts 65+

20 min Physical Measurements -

Form 80 (other than height &

weight) Resting pulse, blood

pressure

10 min Time 3 – Spot urine collection

and processing: 3 hours after

DLW

~ 60 min

5 min Time 3 - Blood collection sample

(non-fasting) - age 60 or older

20 min Review and scan Form 35 and

Form 60. Clarify missing

information with participant.

Review Form 33

Complete Form 33D, as

needed

10 min CaD - Complete adherence

to study pills

5 min CaD - Complete

management and safety

interview (Form 17)

5 min CaD - Dispense study pills

5 min Exit interview

10 min Time 4 – Spot urine collection

and processing - 4 hours after

DLW

NBS Manual Page 20


Figure 5.2 – Integration of NBS Visit 1 and Annual Visit Tasks (continued)


~ 15 min

Estimated

Times


Visit Tasks*

CaD & HT Annual

Visit Tasks*

Year 9 Annual

Visit Tasks*

10 min Provide instructions and kit for

24-hour urine collection -

5 min Confirm appt. for next visit

Provide complementary meal

(e.g., box lunch)

5 min Daily Life – 6% sub sample

30 min ECG

30 min Bone density sites only -

bone density scan

NBS Manual Page 21


5.1.3 Pre-NBS Visit 1 Activities

5.1.3.1 Supplies and Forms

NBS staff confirm that all supplies and forms needed for NBS Visit 1 are available for the participant’s

visit.

Supplies:

WHI Folder (for participant handouts)

NBS label set

Urine collection hat

Doubly labeled water (DLW)

Lavender (dry EDTA) blood collection tube (for participants > 60 years of age)

Corning cryovials, 5 mL

2.0 mL cryovials

Disposable transfer pipettes

Ziplock bag with pre-weighed tissue

Meal replacement beverage (e.g., Boost, Ensure)

24-hour urine collection kit (Instructions for 24-hour Urine Collection, urine collection hat, 2 [3-

liter] containers, plastic funnel, large safety pin, and 2 gel ice packs in a plastic carrying bag)

- For participants who receive PABA, the kit contains:

o A highlighted copy of the Instructions for 24-hour Urine Collection where there is a

reminder to “take one PABA (B-vitamin) tablet with each meal” on the day of the

urine collection (use a Hi-Liter marker to call attention to this text).

o 3 sealed PABA (B-vitamin) tablets

o 2 [3-liter] urine collection bottles in each of which you place 2.0 gram (1.5 level

measuring teaspoons) boric acid * and on each of which you place two stickers, one

that reads “Leave powder preservative inside bottle” and one that reads

“Remember…take the PABA (B-vitamin) tablets”

* The boric acid may be weighed or measured using household measuring spoons

labeled, “For boric acid use only.” When handling the boric acid, wear disposable

gloves and preferably a disposable mask. Even though boric acid is a safe

preservative, take precautions to avoid skin contact or inhalation.

Complementary meal

Forms:


NBS Consent

Form 75 - NBS Visit 1

Form 60 – Food Frequency Questionnaire

Form 35 – Personal Habits Update

Form 45 – Current Supplements (Backup) - (have available if not directly key-entering

supplement information into WHILMA using Task 45)


5.1.3.2 Pre-Visit 1 Reminder Call

A NBS staff person calls each participant one day prior to her NBS Visit 1. If a participant is scheduled

for Monday, the call may be made on the preceding Friday. During the call, complete the following:

Confirm the participant’s NBS Visit 1 appointment.

NBS Manual Page 22


Remind the participant to refrain from eating any food or drinking any caffeine or calorie-

containing beverages for 4 hours prior to her clinic appointment time.

Let participant know that during the 4-hour fast she may take all her regular medications with

water and she may drink decaffeinated black coffee or herbal tea (without milk, cream or sugar).

Regular (caffeinated) coffee or tea should be avoided. Encourage the participant to drink water

liberally during the fast; otherwise, she may become dehydrated which can be uncomfortable for

the participant and result in difficulties collecting urine and blood samples.

Ask the participant to bring all her dietary supplements to the visit (they will be documented in

Task 45 – Current Supplements).

Ask the participant to wear clothing which allows the sleeve to be easily raised above the elbow

without constricting the blood flow to the forearm and hands and allows for ease of multiple urine

collections.

NBS Manual Page 23


5.1.4 NBS Visit 1 Activities

5.1.4.1 Reception

When a participant first arrives at the CC for her NBS Visit 1/AV, have her check-in at the reception

desk. The Receptionist should:

Locate the participant’s file.

Indicate a comfortable place where the participant may wait until she can be seen.

Immediately notify the NBS Lead-ops (or designee) that the participant is waiting.

If a participant arrives at the clinic for her AV without having been reached for the NBS Telephone

Screening Questionnaire/Script and inquires about the NBS study, the Receptionist should immediately

notify the NBS-Lead-ops (or designee) that the participant is interested in NBS. The Lead-ops (or

designee) checks to see if the participant is on the NBS Recruitment Tracking report (NBS001). If yes,

the Lead-ops (or designee) conducts the NBS Telephone Screening Questionnaire/Script to determine

interest and eligibility. If eligible, the Lead ops (or designee) uses the procedures outlined in the NBS

Manual, Section 4.1.3 – Schedule NBS Visit 1 and Section 4.1.4 – Complete and Data Enter NBS

Screening Result (Form 74).

5.1.4.2 Eligibility

Some of the NBS eligibility questions asked during the telephone screening call are repeated at NBS

Visit 1 to ensure that the participant remains eligible. Begin the participant’s visit by using the NBS

Visit 1 Eligibility Checklist Worksheet to update a participant’s continuing eligibility.

If a participant reports any change in the information she previously provided on the NBS

Telephone Screening Questionnaire/Script that now make her ineligible (e.g., began taking

insulin or oral hypoglycemics), mark her “Ineligible” on Form 75 – NBS Visit 1. Thank the

participant and escort her to the WHI staff person to start the WHI AV tasks. File the NBS Visit 1

Eligibility Checklist Worksheet in the participant’s chart.

If a participant reports a change that would make her ‘temporarily ineligible’ (e.g., traveled more

than 200 miles in past 2 weeks) for NBS Visit 1 and she is not willing to reschedule her NBS

Visit 1, mark her “Ineligible” on Form 75 – NBS Visit 1. Thank the participant and escort her to

the WHI staff person to start the WHI AV tasks. File the NBS Visit 1 Eligibility Checklist

Worksheet in the participant’s chart.

If a participant reports a change that would make her ‘temporarily ineligible’ (e.g., traveled more

than 200 miles in past 2 weeks) for NBS Visit 1, but she is willing to reschedule her NBS Visit 1,

thank the participant, reschedule her NBS Visit 1 for another day and then escort her to the WHI

staff person to start the WHI AV tasks. File the NBS Visit 1 Eligibility Checklist Worksheet in the

participant’s chart.

If a participant remains eligible, mark “Eligible” on Form 75 – NBS Visit 1 and begin the NBS Consent

procedures.

5.1.4.3 NBS Consent

Refer to NBS Manual, Appendix B – NBS Consents and the Appendix for Section 10 – Indirect

Calorimetry, for a copy of the appropriate NBS Consent.

Mailing the NBS Consent Before NBS Visit 1. The CCC recommends that the NBS Consent not be sent

with the NBS invitation letter. It could be overwhelming to the participant to read the NBS Consent

without having had the opportunity to discuss the NBS protocol with staff. If a CC wants to send the

NBS Consent to interested participants, the CC could mail the consent to the participant after she has

completed the NBS phone screening contact and is scheduled for NBS Visit 1.

NBS Manual Page 24


Reviewing the NBS Consent. Provide the participant with a copy of the NBS Consent. Review the

consent with the participant after she has had ample time to read it. Ask her if she has any questions and

answer her questions thoroughly. The following key points must be covered with the participant:

Her participation in the Nutritional Biomarkers Study (NBS) is voluntary and does not affect her

participation in the WHI Dietary Study. She may withdraw from NBS at any time.

Any information she gives will be kept completely confidential and will be released to no one

except WHI personnel and, if appropriate, authorized NHLBI staff.

Her responses will be added to those of other participants and only grouped information will be

released. Neither her name nor any other identifying information will be released in WHI reports

or publications.

A description of the NBS procedures during Visit 1 and Visit 2 (e.g., height, weight,

administration of DLW, spot urines and blood draws).

A description of each procedure and any risks associated.

The loading dose of doubly labeled water may cause temporary vertigo. This is rare at

the small tracer dose used for this study. As a precaution, however, participants should

sit down when drinking the loading dose and should remain seated for 15 minutes after

drinking the loading dose.

The blood draw may cause discomfort and a bruise at the site of the needle puncture.

All efforts will be made to minimize this risk.

People with known allergies to PABA may get a slight rash if they take the PABA

vitamin. Women who know they are allergic to PABA will not be asked to take it.

The participant may find it inconvenient to collect urine for a 24-hour period.

The participant will receive $100 after the second visit for time and travel expenses. Participants

who do not return for the second visit procedures will not receive the $100.

The potential of repeating the entire study about six months later to help us learn about the

reliability of the measurements. Participants who repeat the entire study will also be asked to

complete two 20-minute dietary phone interviews (24-hour recalls).

Each CC must also follow the requirements imposed by their own Institutional Review Board (IRB) in

carrying out the informed consent procedures.

Signing the NBS Consent:

Once the participant’s questions have been answered, ask her to sign and date two copies of the

NBS Consent in the appropriate places. Sign two copies of the form yourself, as a WHI

representative, and witness and date the form. Give one copy of the NBS Consent to the

participant and file the other copy in her file. Record on Form 75 – NBS Visit 1 that the

participant has signed the NBS Consent.

If a participant declines to sign the NBS Consent, do not continue further NBS activities. Record

on Form 75 – NBS Visit 1 that the participant did not sign the NBS Consent. Thank the

participant, end the NBS visit and have the participant continue with her regularly scheduled AV

activities.

Provide a WHI Folder: Provide each participant with a WHI folder to hold the NBS study materials

provided at NBS Visit 1 (i.e., copy of NBS Consent, Study At A Glance and Instructions for 24-hour

Urine Collection).

5.1.4.4 Provide Study at a Glance Handout

Give participants, who consent to participate in NBS, a copy of Study At A Glance (copy available in

NBS Manual, Appendix C – NBS Participant Materials). This handout provides a brief overview of the

activities that each participant will do as part of the Nutritional Biomarkers Study.

NBS Manual Page 25


5.1.4.5 Physical Measurements (height and weight)

Collect the height and weight on the day of the NBS Visit. Use a measured height and weight (not an

estimate). A measured height and weight are critical to correctly estimate total energy expenditure from

the doubly labeled water and for the statistical modeling planned for the NBS study.

Record the height and weight in centimeters and kilograms, respectively, on Form 75 - NBS Visit 1.

Staff can copy the participant’s NBS height and weight information onto her AV Form 80 – Physical

Measures (i.e., once the participant’s height and weight have been measured for NBS, they do not need

to be re-measured for her AV).

5.1.4.6 Spot Urine Collection – NBS Visit 1

5.1.4.6.1 General Information

There are four spot urine samples collected at NBS Visit 1: a fasting sample (before the doubly labeled

water [DLW] is administered) and three samples post DLW administration at approximately 2, 3, and 4

hours.

Refer to NBS Manual, Section 7.2.2.2.1 – Timing of the Spot Urine Collections and Section 7.2.2.2.2 –

DLW Spot Urine Collection Steps for information about the collection and processing of the spot urine

samples at NBS Visit 1.

Thoroughly Dry Collection Devices The spot urine collections will be done using a urine collection hat (the hat is placed on the toilet, under

the toilet seat). Each participant will have one urine collection hat for all her spot urine collections at

NBS Visit 1. The urine hat will need to be rinsed and thoroughly dried with a paper towel between each

spot urine. It is critical that the collection device be DRY before each urine collection because any

water that contaminates the urine either during collection or processing will dilute the isotopic

concentration of the DLW tracers.

Prevent Evaporation Do not leave the urine open to the air so that it can either evaporate or exchange with the moisture of the

air. A minute or two is okay, but do not leave the urine sitting around for 15 minutes without being

capped. Ideally, have the participant’s NBS label on the cryovial; transfer the urine to the 5 mL

cryovial, cap and store within 10 minutes. Refer to NBS Manual, Section 7.2.2.2.1 – Timing of the Spot

Urine Collections and Section 7.2.2.2.2 –DLW Spot Urine Collection Steps for information about the

collection and processing of the spot urine samples at NBS Visit 1.

5.1.4.6.2 Time 0 (Fasting) Urine Collection

The Time 0 (fasting) spot urine needs to be collected, as soon as possible, after the participant’s height

and weight measurements have been taken. The fasting urine does not have to be the participant’s first

urine of the day BUT the urine must be collected BEFORE the participant drinks the DLW because the

tracer gets in the urine very quickly (within 30 seconds). When the sample is collected, record staff ID

(staff collecting urine) and time of collection on Form 75- NBS Visit 1.

Use the procedures described in the NBS Manual, Section 7.2.2.2.1 – Timing of the Spot Urine

Collections and Section 7.2.2.2.2 –DLW Spot Urine Collection Steps for information about the


NBS Manual Page 26


5.1.4.7 Administration of Doubly Labeled Water (DLW) – Fasting

5.1.4.7.1 DLW Doses

Clinics will receive pre-filled bottles with doubly labeled water. The DLW doses are meant to provide

enough of the tracer to the participant so that it can be measured easily, but not a huge dose, because the

DLW is expensive. There are four dose sizes (A, B, C and D). These are designed for individuals who

are respectively:

A – Less than 65 kg

B – 66 to 80 kg

C – 81-105 kg

D – Greater than or equal to 106kg

5.1.4.7.2 DLW Administration

Select the DLW bottle appropriate for the participant’s weight. If a participant is right on the cusp

between weights, staff can go either way (e.g., if the participant is 80kg, staff could give her either a B

or a C bottle). If a scheduled participant should be getting a B bottle, but the clinic is out of B bottles,

staff may use an A or a C bottle (one bottle size either direction is okay). Contact Helen Penor

([email protected]) at the CCC if the CC is running out of a specific-sized bottle.

Record staff ID (staff administering DLW), the 7-digit dose weight of the bottle and the lot number of

the bottle on Form 75- NBS Visit 1. The doses vary slightly, so it is critical to get the correct

information recorded on the form.

Give the participant a straw and ask her to drink all the DLW (about ½ cup). If the participant would

prefer to drink directly from the bottle, she may as long as her hands are steady enough to hold the bottle

when she drinks.

Record the time the participant begins drinking the DLW dose on Form 75- NBS Visit 1.

After the participant drinks the contents of the DLW bottle, there is usually about half a gram of labeled

water still left in the bottle. To guarantee that the participant has received the entire DLW tracer, fill the

DLW dose bottle with 50 mL tap water and push the straw into the bottle. Replace the cap and gently

mix the water around so that it captures all the labeled water on the cap, inside of the bottle, and the

straw. Give the participant a second straw and ask her to drink the rinse water. Note: the amount of the

rinse water (50 mL) does not need to be recorded on Form 75 – NBS Visit 1.

Ask the participant to remain seated for about 15 minutes after drinking the DLW to minimize the small

chance of dizziness that may occur upon drinking the loading dose.

5.1.4.7.3 Handling DLW Spillage

Each CC will receive a supply of pre-weighed Kleenex tissues, each in a ziplock bag. The weight will

be written on the bag.

If there is a small spill where staff can mop it up (on the participant’s cheek, on the floor, etc.), use the

pre-weighed tissue to mop up the spilled DLW.

Immediately return the tissue to the ziplock bag and seal it. Find the ‘NBS DLW Spillage Container

Visit 1’ label on the participant’s NBS label set.

Place the participant’s ‘NBS DLW Spillage Container Visit 1’ label on the bag and date the bag.

Record ‘Yes’ for DLW spillage on the Form 75- NBS Visit 1 (Qx. 13.5).


NBS Manual Page 27


The same day, Fed-Ex the bagged tissue to Dr. Dale Schoeller at address below:

Dale A. Schoeller

Nutritional Sciences

University of Wisconsin

1415 Linden Drive

Madison, WI 53706

Phone #: 608-262-1082 FAX: 608-262-5860

5.1.4.7.4 Storage of DLW

The DLW bottles are prepared in a sterile manner and may be stored on a shelf at room temperature.

The bottles do not need to be refrigerated. Keep them out of the sunlight, so that they don’t get hot.

While the bottles do not need to be refrigerated, the water tastes better chilled. The plastic bottles tend

to give the water a bit of a ‘plastic’ taste, but chilling the water tends to make it a little more palatable.

Suggestion: chill the bottles overnight before giving them to a NBS participant to drink.

5.1.4.8 Provide WHI Forms 35 & 60 and Complete Task 45

Give the participant Form 35 (Personal Habits Update) and Form 60 (FFQ) to complete. Ask her to

complete these forms while she is waiting between visit activities. Let the participant know that a staff

person will review both forms with her before the end of her visit.

Complete Task 45 - Current Supplements

The purpose of collecting an inventory of current supplements at NBS Visit 1 is to document the

supplements a participant takes that could influence the levels of vitamins and minerals in her

blood.

Enter each current vitamin and mineral supplement into WHILMA following the procedures

described in WHI Manuals, Vol.5 – Data System, Section 7.3.2 – Current Supplements. Remind

the participant to report only current supplements taken at least once a week. The supplement

inventory program will offer specific prompts for entering the vitamin formulations when needed.

Use Form 45 – Current Supplements (Backup) for completing the supplements inventory if the

computer is not available.

If the participant has forgotten to bring her supplements, arrange to have her bring them to NBS

Visit 2 or call her at home to follow-up.

If a participant is completing Task 45 - Current Supplements as part of her AV, record ‘3 –

Annual’ in the “Visit Type” field on the form and indicate the appropriate year. If a participant is

not completing Task 45 as part of her AV visit, record as ‘4 – Non Routine’ in the “Visit Type”

field.

5.1.4.9 Provide Meal Replacement Beverage (MRB)

One hour after administration of the doubly labeled water (DLW), provide the participant with an 8-

ounce (240 mL) meal replacement beverage (e.g., Boost, Ensure, etc.) or other beverage of choice.

Record “Yes” on Form 75, Qx. 14.1 – MRB Consumed and then record the amount (mL) and time the

participant begins drinking the MRB (or other beverage). Encourage the participant to drink the

beverage in a timely manner, rather than sipping.

5.1.4.10 Two Hour Urine Collection

Two hours post DLW dose, have the participant provide a second spot urine sample. Use the

procedures described in the NBS Manual, Section 7.2.2.2.1 – Timing of the Spot Urine Collections and

NBS Manual Page 28


Section 7.2.2.2.2 –DLW Spot Urine Collection Steps for information about the collection and processing

of the spot urine samples at NBS Visit 1.

Record staff ID (for staff collecting the urine) and time of collection on Form 75 – NBS Visit 1.

If a participant has problems with the void, follow the procedures for handling collection problems,

described in the NBS Manual, Section 5.1.4.10.1 – Handling Collection Problems.

5.1.4.10.1 Handling Collection Problems

Participant Produces No Urine

If a participant is unable to produce urine for the 2nd

, 3rd

or 4th

spot urine collection, give her a little

something to drink (i.e., ½ cup of water) and then have her try again in about half an hour. Record the

amount and time of fluid intake on Form 75 – NBS Visit 1, (Qx. 18 - Other Beverages Consumed).

After a half-hour, if a participant is still unable to provide a spot urine sample, run warm water

over the inner part of the participant’s wrists. If still unable to provide a spot urine sample leave

the appropriate urine collection question blank and indicate in the “Notes” section at the end of

Form 75-NBS Visit 1 that the participant was unable to provide a Time X urine specimen. Follow

the procedures outlined in the NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify the

CCC.

Participant Produces Very Little Urine If a participant produces very little urine for the 2

nd, 3

rd or 4

th spot urine collection (less than 10 mL of

urine), toss out the urine collection, give her a little something to drink and have her try again in about a

half-hour. Record the amount and time of fluid intake on Form 75 – NBS Visit 1, (Qx. 18 - Other

Beverages Consumed). If still unable to produce a urine sample, run warm water over the inner part of

the participant’s wrists. Note: Do not combine urine collections together to create a larger volume (i.e.

combining first attempt with second attempt).

5.1.4.11 Three Hour Urine Collection

Three hours post DLW dose, have the participant provide a third spot urine sample. Use the procedures

described in the NBS Manual, Section 7.2.2.2.1 – Timing of the Spot Urine Collections and Section

7.2.2.2.2 –DLW Spot Urine Collection Steps for information about the collection and processing of the

spot urine samples at NBS Visit 1.

Record staff ID (staff collecting the urine) and time of collection on Form 75 - NBS Visit 1.



5.1.4.12 Three Hour Blood Draw (for women > 60)

For women 60 years of age and older, there is often an increase in the prevalence of post-void residual

volume in the bladder. This means that the bladder does not totally empty out and this indirectly creates

a problem in calculating energy expenditure. To help resolve this problem, a blood sample is used to

check against the urine volume to detect if there was post-void residual volume.

Three hours post DLW dose, draw a blood sample from women who are 60 years of age or older. The

3-hour blood draw must occur just after the 3-hour spot urine collection. If the 3-hour spot urine is

slightly delayed, the 3-hour blood draw should also be delayed to immediately after that urine

collection. Record the blood draw on Form 75 - NBS Visit 1. Use standard WHI blood handling and

processing procedures described in the NBS Manual, Section 7.2.2.2.6 – Time 3 Blood Draw (3 hours

after DLW ingestion, for women 60 years of age or older).

NBS Manual Page 29


If a participant is under 60 years of age, place a note on the top of Form 75 –NBS Visit 1 next to the

participant’s member ID label – “Participant is under 60”. This will help NBS staff easily identify

participants who do not need the 3-hour blood draw.

If a participant has problems with the blood draw, follow the procedures for blood collection problems,

described in the NBS Manual, Section 7.4.4 – Blood Collection Problems.

5.1.4.13 Collect and Review Form 35 and Form 60

Form 35 (Personal Habits Update) and Form 60 (FFQ) are to be completed, reviewed and scanned

before the participant leaves the clinic. Whenever possible, use staff blinded to DM assignment to

review the forms.

Make sure that there is a WHI participant bar code label in the designated space on the front page of

each completed Form 35 and Form 60. (Refer to WHI Manuals, Vol. 3 – Forms for specific instructions

regarding the items on each form).

If a participant is completing a Form 35 (Personal Habits Update) or Form 60 (FFQ) as part of their

AV, record ‘3 – Annual’ in the “Visit Type” field on the form and indicate the appropriate year. For

participants who are not completing a Form 35 or Form 60 as part of their AV visit, record ‘4 – Non

Routine’ in the “Visit Type” field on the form.

a) Form 35 – Personal Habits Update

Information about the participant’s physical activity will be captured using

Form 35 (Personal Habits Update).

b) Form 60 – Food Frequency Questionnaire

Information about the participant’s dietary intake will be captured using

Form 60 (FFQ).

Probe for additional information about missing answers. All adjustment questions (pages 2-4)

and all summary questions (page 12) on the FFQ must be answered. Refer to WHI Manuals,

Vol. 3 – Forms for more details about reviewing and editing the FFQ.

5.1.4.14 Four Hour Urine Collection

Four hours post DLW dose, have the participant provide a fourth spot urine sample. Use the procedures



spot urine samples at NBS Visit 1.

Record staff ID (staff collecting the urine) and time of collection on Form 75 - NBS Visit 1.



5.1.4.15 Other Beverages

It is important to record all the fluids that a participant drinks. Participants should not have free access

to fluids – intake needs to be controlled. Record all fluid intake between the time of the DLW dose and

the last spot urine sample at NBS Visit 1. Strongly encourage participants to drink fluids in a timely

manner, rather than sipping.

NBS Manual Page 30


Participants should preferably have no more than 250 mL of fluids in addition to the 250 mL in the meal

replacement beverage (MRB) for a total of 500 mL. But, it is important to get the urine specimens, so if

a participant needs to go slightly above this 500 mL total to get the 3 or 4 - hour spot urine (e.g., the

participant is dried out), staff may go ahead and provide a small amount of fluid. However, one liter is

the upper limit; do not go above the one liter limit.

Participant may have either caloric or non-caloric beverages, such as coffee, tea, or juice. These

beverages do not interfere with the collection of the post-DLW protocol.

When measuring the amount of other beverages, it is not necessary to measure the beverage in a

graduated cylinder. A measurement within 10-20 mL of actual volume is close enough. Record the

amount and time of beverage consumption on Form 75 - NBS Visit 1 (Qx. 18 - Other Beverages

Consumed)

If a participant does not drink any other beverages between hours 2-4 after the DLW, record ‘0’ for Qx.

18.1 (Beverage 1 Amount) and Qx. 18.2 (Beverage 2 Amount) on Form 75 - NBS Visit1.

5.1.4.16 Explain and Provide Materials for 24-hour Urine Collection

Near the end of NBS Visit 1, explain to the participant that she will need to collect all of her urine for 24

hours. She will begin her 24-hour urine collection on the day before her NBS Visit 2 (Day 14) and

bring it back to her NBS Visit 2 appointment (Day 15). The 24-hour urine collection will be used to

determine urinary nitrogen (a marker of protein intake).

The 24-hour urine collection is preferably scheduled to coincide with the collection being completed for

NBS Visit 2 on Day 15. However, for participant convenience, NBS Visit 2 may be scheduled for Day

14 or Day 16.

Review the information provided on the Instructions for 24-hour Urine Collection with the participant

(copy located in the NBS Manual, Appendix C – NBS Participant Materials). Provide clarification as

needed.

PABA

The PABA procedure helps to determine completeness of the 24-hour urine collections. PABA is a type

of B-vitamin that is absorbed but not metabolized by humans. A few participants may be allergic or

hypersensitive to PABA and should not receive it.

Ask participants if they ever had hypersensitivity reactions to PABA-containing sunscreens or are

allergic to PABA.

o If yes (allergic to PABA):

Remove the PABA (B-vitamin) tablets from the urine collection kit and provide urine

collection bottles that do not have boric acid inside.

Record in the “Notes” section of Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3)

that the participant was not given the PABA (B-vitamin) tablets because she has

had past hypersensitivity to PABA. Follow the procedures outlined in NBS Manual,

Section 8.2.1 – Notes on NBS Forms to notify the CCC

o If no (not allergic to PABA):

Instruct participants to take one PABA (B-vitamin) tablet with each meal (or one after

the first urine is flushed, one mid-day, and one in the evening). Point participants to

the text on the Instructions for 24-hour Urine Collection where there is a reminder to

“take one PABA (B-vitamin) tablet with each meal” on the day of the urine collection

(use a Hi-Liter marker to call attention to this text).

Instruct participants to refrain from taking acetaminophen (e.g., Tylenol) or vitamins

when collecting the 24-hour urine sample.

Ask participants to bring back the PABA tablet packaging with her to NBS Visit 2.

NBS Manual Page 31


Attach a WHI participant member ID to a NBS Visit 2 (or Visit 4) PABA Worksheet for Staff and mark

the appropriate response for Question 1 (Did the 24-hour urine kit contain the 3 PABA (B-vitamin)

tablets?). Place the NBS Visit 2 (or Visit 4) PABA Worksheet for Staff in the participant’s chart (or

special NBS notebook) so that it will available for use at NBS Visit 2 (or Visit 4).

Provide the participant with a carrying bag containing the following 24-hour urine collection supplies:

Instructions for 24-hour Urine Collection, includes a Record Sheet for 24-hour Urine Collection

(last page of instructions). For participants who receive PABA, substitute a highlighted copy of

the Instructions for 24-hour Urine Collection where there is a reminder to “take one PABA (B-

vitamin) tablet with each meal” on the day of the urine collection (use a Hi-Liter marker to call

attention to this text).

Two 3 liter urine collection bottles. For participants who receive PABA, substitute 2 [3-liter]

containers that have 2.0 gram (1.5 level measuring teaspoons) boric acid inside each bottle and

are labeled with two stickers that read “Leave powder preservative inside bottle” and

“Remember…take the PABA (B-vitamin) tablets”

One urine collection hat

One plastic funnel

One large safety pin

Two gel ice packs

One plastic carrying bag

For participants who receive PABA, the kit additionally contains 3 sealed PABA (B-vitamin)

tablets.

Inform the participant that a NBS staff person will call to remind her when to begin her 24-hour urine

collection. Explain to the participant that she will need to bring her 24-hour urine collection, the

Record Sheet for 24-hour Urine Collection and the gel ice packs to her NBS Visit 2. For participants

who receive PABA (B-vitamin) tablets, explain that she will also be asked to bring the PABA tablet

packaging with her to NBS Visit 2.

5.1.4.17 Confirm Appointment for NBS Visit 2

The NBS Visit 2 occurs on Day 15, fourteen days after NBS Visit 1.

Confirm that the participant has a NBS Visit 2 appointment scheduled for a date that is two weeks after

the first NBS visit. Schedule a NBS Visit 2 appointment, if necessary. If needed, NBS Visit 2 could be

scheduled one day before or after Day 15.

5.1.4.18 Provide Complementary Meal

Thank the participant for her participation and provide a complementary meal (e.g., box lunch). The

meal can be offered anytime after her last spot urine sample (i.e., 4-hour spot urine).

5.1.4.19 Check Visit 1 Completeness

Use the Completeness Checklist on the last page of Form 75- NBS Visit 1 to check that all NBS Visit 1

activities have been completed. Complete any remaining tasks, if required. The staff member who

reviews Form 75 – NBS Visit 1 and checks completion of NBS Visit 1 tasks, designates completion by

recording their Staff ID on Form 75- NBS Visit 1 (Qx. 20 – Visit 1 Completeness Staff ID).

NBS Manual Page 32


5.1.5 Post-NBS Visit 1 Activities

5.1.5.1 Follow-up on Missing Forms

CCs should have a data back-up plan if problems arise with collection of Form 35 (Personal Habits

Update) and Form 60 (FFQ). If a participant leaves the clinic and NBS staff discover that Form 35 –or

Form 60 are missing or have incomplete data, assign a staff person to follow-up with the participant

within 1-2 days of NBS Visit 1.

5.1.5.2 Data Entry

Key-enter Form 75 - NBS Visit 1 for all participants within 1-2 days of NBS Visit 1. File the completed

and key-entered Form 75 - NBS Visit 1 in the participant’s chart (or special NBS notebook).

NBS Manual Page 33


5.2 NBS Visit 2 (Day 15)

5.2.1 Overview of Activities

Figure 5.3 – Overview of NBS Visit 2 (Day 15) provides an overview of NBS Visit 2.

Figure 5.3

Overview of NBS Visit 2 (Day 15)

Participant Arrival

Greet participant

Complete NBS Visit 2 Participant Update Worksheet

Receive 24-hour urine collection

Obtain weight

Obtain and process Time 0 (fasting) spot urine sample

Obtain and process Time 0 (fasting) blood draw

Provide snack/meal

Obtain and process Time 1 spot urine

(1-hour post fasting urine)

Discuss repeat of NBS protocol in about 6 months. Complete

Consent for Future Contact and Continued Participation in this

Study

Schedule NBS Visit 3 appointment, if participant consents

to repeat

Begin necessary institutional paperwork for participant’s

$100 for time and travel

Thank participant for completing the NBS protocol

Key enter Form 76 - NBS Visit 2 and the information from

the NBS Visit 2 (or Visit 4) PABA Worksheet for Staff

NBS Manual Page 34


5.2.2 Pre-NBS Visit 2 Activities

5.2.2.1 Supplies and Forms

NBS staff confirm that all supplies and forms needed for NBS Visit 2 are available for the participant’s

visit.

Supplies:

NBS label set (must be the remainder of the label set started at NBS Visit 1

for the specific participant)

Royal blue and lavender (dry EDTA) blood collection tubes

Corning cyrovials, 5mL

2.0 mL cyrovials

Graduated cylinder to measure 24-hour urine

Urine collection hat

Disposable transfer pipettes

Snack

Forms:


Form 76 - NBS Visit 2

NBS Consent for Future Contact and Continued Participation in this Study

NBS Visit 2 (or Visit 4) PABA Worksheet for Staff (should be in participant’s file from Visit 1 [or

Visit 3])

5.2.2.2 Pre-Visit 2 Reminder Call

Two days prior to the participant’s scheduled NBS Visit 2, call the participant. Remind her to start the

24-hour urine collection by collecting all of her urine beginning at 8 AM the next day using the

specially provided urine collection kit.

5.2.2.2.1 Review Instructions for 24-hour Urine Collection (Day 14)

Review the steps the participant needs to take to complete her 24-hour urine collection. Ask her if she

has any questions and answer her questions thoroughly. The following key points must be covered with

the participant:

The collection period begins at 8:00 AM on the morning of the DAY BEFORE the participant’s

NBS Visit 2. The collection ends at 8:00 AM on the morning of the DAY OF her appointment.

The time can vary slightly as long as the collection time is 24 hours. If her appointment is

scheduled early in the morning, she may start and end earlier than 8:00 AM.

During the at-home collection, containers containing urine should be tightly capped and stored in

a refrigerator on the bottom shelf at least 6 inches away from food. It may also be stored in a

cooler with ice if preferred.

During the urine collection period:

1. The participant begins by emptying her bladder at 8:00 AM.

2. She is to flush this urine. She will collect all urine after this point but record this time as

“Start of collection” on the attached record sheet (Record Sheet for 24-hour Urine Collection).

3. If the urine kit contains the PABA (B-vitamin) tablets:

She should take one with each meal (or one after the first urine is flushed, one mid-day, and

one in the evening). She is not to take acetaminophen (e.g., Tylenol) or vitamins during the

collection period. If she has had hypersensitivity reactions to PABA in sunscreens, she should

not have PABA (B-vitamin) tablets in her urine kit.

NBS Manual Page 35


4. She is to attach the large safety pin to her underclothing to serve as a reminder that she is

collecting urine. It should be in a place that she can easily see when she goes to the

restroom.

5. The next time she goes to the restroom, she is to place the urine collection hat in the toilet

(under the toilet seat) before urinating. It is important that she collects all of the urine. If

she needs to have a bowel movement, she should collect the urine separately.

6. After urinating, she is to use the plastic funnel to pour the urine from the urine collection hat

into the collection container. Let the participant know that she should completely fill one

urine bottle before starting to fill a second container.

7. She is to continue collecting all urine for 24 hours (all day and night.).

8. At 8:00 AM (or 24 hours from her START time) the following day, the NBS participant is to

empty her bladder into the urine collection hat and add this final urine to the collection

container. The exact time of this last collection should be recorded on the “End of

Collection” line on the Record Sheet for 24-hour Urine Collection.

9. The participant is to note any problems with the urine collections on the Record Sheet for 24-

hour Urine Collection (last page of Instructions for 24-hour Urine Collection). A copy of

the instructions is available in the NBS Manual, Appendix C – NBS Participant Materials.

10. The participant is to bring all urine collected, the Record Sheet for 24-hour Urine Collection

and the gel ice packs with her to NBS Visit 2.

If the participant received PABA (B-vitamin) tablets, she is to bring back the PABA tablet

packaging with her to NBS Visit 2.

5.2.2.2.2 Confirm Appointment and Fasting Time

Confirm the participant’s NBS Visit 2 appointment. If the participant needs to re-schedule her clinic

appointment (e.g., emergency situations), try to reschedule the appointment within + one day. Do not

go longer than 20 days post NBS Visit 1.

Remind the participant to:

Refrain from eating any food for 6 hours prior to her clinic appointment. Encourage the

participant to drink water liberally during the fast; otherwise, she may become dehydrated which

can be uncomfortable for the participant and result in difficulties collecting urine and blood

samples. Black coffee or tea, without milk, cream or sugar (caffeinated or decaffeinated) is okay

at NBS Visit 2.

Take all her regular medications with water on the day of NBS Visit 2.

Wear clothing which allows the sleeve to be easily raised above the elbow without constricting

the blood flow to the forearm and hands and allows for ease of multiple urine collections.

Ask the participant if she has questions and provide information as needed.

5.2.3 NBS Visit 2 Activities

5.2.3.1 Reception – Welcome

When the participant first arrives for NBS Visit 2, have her check in at the reception desk. The

Receptionist should:

Locate the participant’s file.

Indicate a comfortable place where the participant may wait until she can be seen.

Notify the NBS Lead-ops (or designee) that the participant is waiting.

NBS Manual Page 36


5.2.3.2 Complete NBS Visit 2 Participant Update Worksheet

Complete NBS Visit 2 Participant Update Worksheet to monitor and note potential changes that might

influence the spot urine samples collected at NBS Visit 2. None of the questions on this worksheet will

make a participant ineligible (i.e., travel, use of IV fluids, etc.). The NBS Lead-ops (or designee)

reviews the NBS Visit 2 Participant Update Worksheet and takes the appropriate actions outlined below.

If a participant marks ‘No’ to questions 1-4 on the NBS Visit 2 Participant Update Worksheet,

NBS staff should:

Record their staff ID on Form 76 – NBS Visit 2, Qx. 5 – Staff ID to Indicate Completion of the

Visit 2 Participant Update Worksheet.

Begin NBS Visit 2 activities.

If a participant marks ‘Yes ’ to question 1, 2 or 3 on the NBS Visit 2 Participant Update

Worksheet, NBS staff should:

Note the participant’s response on the line next to the appropriate question.




At the end of NBS Visit 2, follow the procedures outlined in the NBS Manual, Section 8.2.1

– Notes on NBS Forms to notify the CCC.

If a participant marks ‘No ’to questions 1, 2 and 3, but ‘Yes’ to question 4 on the NBS Visit 2

Participant Update Worksheet (indicating that she has not fasted for 6 hours), NBS staff should:



Refer to the procedures in the NBS Manual, Section 5.2.3.6.1-Handling Non-Fasting

Participants and Section 7.4.4.1 – Directions for Staff When Participants Are Not Fasted to

manage the blood draw for non-fasting participants.


5.2.3.3 Receive 24-hour Urine Collection

Follow procedures outlined in the NBS Manual, Section 7.3.3.2.1 – Receive the 24-hour Urine

Collection from Participant to receive the participant’s 24-hour urine collection, review her Record

Sheet for 24-hour Urine Collection, and complete the NBS Visit 2 (or Visit 4) PABA Worksheet for Staff.

Handling 24-hour Urine Collection Problems:

If a participant arrives at the CC with an incomplete or contaminated 24-hour urine collection, the

Lead-ops (or designee) follows the procedures outlined in the NBS Manual, Section 8.2.1 – Notes

on NBS Forms to notify the CCC.

If a participant arrives at the CC without a 24-hour urine collection, the Lead-ops (or designee)

decides how to handle the missing 24-hour urine collection using the scenarios provided below:

Participant Forgot to Collect: If a participant forgot to collect her 24-hour urine

sample, reassign a day for the collection within 3 working days. Continue with the

remaining NBS Visit 2 activities.

Participant Forgot Collection at Home: If a participant completed her 24-hour urine

collection but forgot the container(s) at home, ask her to return her 24-hour collection to

the clinic as soon as possible, within 2 working days. Continue with the remaining NBS

Visit 2 activities.

NBS Manual Page 37


Participant Refuses to Collect 24-hour Urine: If a participant refuses to complete her

24-hour urine collection, the NBS Lead-ops (or designee) continues with the remaining

NBS Visit 2 activities and at the end of the visit, follows the procedures outlined in the

NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify the CCC.

5.2.3.4 Physical Measurements (weight)

Measure the participant’s weight in kilograms and record on Form 76 - NBS Visit 2.

5.2.3.5 Spot Urine Collection - NBS Visit 2

5.2.3.5.1 General Information

There are two spot urine samples collected at NBS Visit 2: a fasting sample and a sample one hour later.

Use the procedures described in the NBS Manual, Section 7.2.2.2.1 – Timing of the Spot Urine

Collections and Section 7.2.2.2.2 –DLW Spot Urine Collection Steps for information about the


Use the guidelines provided in the NBS Manual, Section 5.1.4.6.1 - General Information to thoroughly

dry collection devices and avoid leaving the urine sample open to evaporation.


The Time 0 (fasting) spot urine sample needs to be collected, as soon as possible, after completing NBS

Visit 2 Participant Update Worksheet and obtaining the participant’s weight. Use the procedures



spot urine sample at NBS Visit 2.

When the sample is collected, record staff ID (for staff collecting the urine) and time of collection on

Form 76 - NBS Visit 2.

5.2.3.6 Fasting Blood

A fasting blood sample is required for all participants at NBS Visit 2. Draw the participant’s blood if

she has been fasting for at least 6 hours. Use standard WHI blood handling and processing procedures

described in the NBS Manual, Section 7.3.3.2.3 – Time 0 (Fasting) Blood Draw.

Be sure to protect all the blood samples collected at NBS Visit 2 from natural and fluorescent light. For

example, cover vacutainers with foil until processing is completed (refer to NBS Manual, Section

7.3.3.2.3 – Time 0 (Fasting) Blood Draw).

5.2.3.6.1 Handling Non-Fasting Participants

For managing participants who arrive at NBS Visit 2 non-fasting, refer to NBS Manual, Section 7.4.4.1

– Directions for Staff When Participants Are Not Fasted.

5.2.3.7 Snack/Meal

After the fasting blood is drawn, give the participant a light snack (e.g., juice, fruit, or crackers) while

she is sitting in a quiet, comfortable place, away from the blood-drawing area. Serve foods that will

help to bring blood sugar back up quickly. Some examples include fruit juices, fruit, granola bars,

crackers, English muffins, bagels or toast (offered with jelly, margarine or peanut butter).

NBS Manual Page 37a


5.2.3.8 One Hour Urine Collection

A final spot urine sample is collected 1 hour after the Time 0 (fasting) sample. Use the procedures



spot urine sample at NBS Visit 2.

When sample is collected, record staff ID (for staff collecting the urine) and time of collection on Form

76- NBS Visit 2.

5.2.3.9 Invite Repeat of NBS - (Reliability Subsample)

After the final spot urine, give the participant the Consent for Future Contact and Continued

Participation in this Study. Refer to NBS Manual, Appendix B – NBS Consents for a copy of the

consent. Explain that this consent is asking the participant if she would be interested in participating in

a repeat of the Nutritional Biomarkers Study in about 6 months.

Ensure that the participant understands that by checking the box “I am willing to participate in a repeat

of the procedures in the WHI Nutritional Biomarkers Study in about six months” – it means that she is

consenting to be part of the reliability subsample (110 women).

Explain that by agreeing to repeat the Nutritional Biomarkers Study, she will be asked to repeat all of

the study procedures she just finished (i.e., drinking doubly labeled water, providing blood and urine

specimens (spot urines at the clinic visits and 24-hour urine collection at home), being weighed, and

completing questionnaires). She will also be asked to complete two telephone interviews about her food

intake - the first about 3 weeks after the NBS Visit 2 and another around the time she repeats the NBS

study in about 6 months. She will also be asked to take a B-vitamin (PABA) the day she collects her

24-hour urine.

There should be at least two months between NBS Visit 2 and the first reliability subsample visit (NBS

Visit 3). Whenever possible, schedule NBS Visit 3 to occur in conjunction with the participant’s WHI

Close-out visit (NBS Visit 3 may be on the same day or a few days before or after the Close-out visit).

In many cases, this scheduling will support the appropriate timing between NBS Visits 1 and 2 and NBS

Visits 3 and 4. On average, there will be approximately 6 months between the WHI AV and the WHI

Close-out visit. However, if a participant’s WHI Close-out visit is scheduled for less than two months

after NBS Visit 2, please choose one of the following options:

Do not offer the participant an opportunity to participate in the reliability subsample.

Schedule the participant’s WHI Close-out visit so that there are at least two months between NBS

Visit 2 and her WHI Close-out visit (and NBS Visit 3), if this is manageable for your CC.

Note: Each CC strives to recruit about 13 participants for the reliability subsample (Tucson and

Memphis strive to recruit about 7 participants). This oversampling helps to ensure that there is an

adequate number of participants available for the reliability subsample. Some participants recruited at

the end of NBS Visit 2 may be unavailable in 6 months when the reliability subsample study begins.

The CCC will monitor response rates for the reliability subsample and let CCs know if they need to

recruit more or fewer than 13 participants for the reliability subsample.

5.2.3.10 Make Arrangements for Time and Travel Costs and Provide Thank You for Participation

Time and Travel Cost Arrangements. When the participant has completed her NBS Visit 2 activities,

initiate the paperwork required by the CC’s local institution to process a check for the participant. The

costs are included in each CC’s total budget.

Thank You. Thank the participant for her participation in the NBS study. If the participant has agreed

to be part of the reliability subsample, let her know that NBS staff will contact her within the next 6

months to conduct two 24-hour recalls.

NBS Manual Page 37b


5.2.4 Post-NBS Visit 2 Activities

5.2.4.1 Data Entry

Key-enter Form 76-NBS Visit 2 for all participants within 1-2 days of NBS Visit 2; include the

information from NBS Visit 2 (or Visit 4) PABA Worksheet for Staff. File Form 76 and NBS Visit 2 (or

Visit 4) PABA Worksheet for Staff in the participant’s chart (or special NBS notebook).

5.2.4.2 24-hour Recalls

For participants who agree to be part of the reliability subsample, the CCC will send each woman a

portion size booklet and a personal letter advising her that staff from the WHI Coordinating Center in

Seattle will contact her approximately 3 weeks after her NBS Visit 2 for the first 24-hour recall. A

second 24-hour recall will be completed around the time that the participant repeats the NBS protocol

about 6 months later.

NBS Manual Page 38


SECTION 6

NBS RELIABILITY SUBSAMPLE

(Information to be added)

NBS Manual Page 39


SECTION 7

BLOOD AND URINE COLLECTION, PROCESSING, AND SHIPMENT

Introduction:

This section of the manual for the WHI Nutritional Biomarkers Study (NBS) describes procedures for collecting,

processing, and shipping blood and urine samples for the WHI NBS.

The NBS protocol calls for three types of specimen collection procedures:

(1) Doubly labeled water (DLW) procedure of spot urine collections (and blood sample for women ≥ 60 years of age)

for the analysis of energy expenditure.

(2) 24-hour urine collection procedure for analysis of (a) urinary nitrogen (UN) as an estimate of protein intake and (b)

select minerals as biomarkers of intake.

(3) Fasting blood draw procedure for analysis of plasma phospholipids fatty acids and biomarkers of micronutrient

intake, e.g., blood tocopherols, retinol, folate, carotenoids, B-vitamins, and selenium.

The three specimen collection procedures occur during two participant in-person clinic visits and one at-home 24-hour

urine collection:

NBS Visit 1

During NBS Visit 1 (or NBS Visit 3), participants provide four spot urine collections for the DLW procedure and,

for women ≥ 60 years of age, one non-fasting blood sample is taken three hours after ingesting the doubly labeled

water. Participants receive instructions and a kit for the 24-hour urine collection procedure that is done one day

before NBS Visit 2.

24-hour Urine Collection

One day before NBS Visit 2 (or NBS Visit 4), participants begin a 24-hour urine collection.

NBS Visit 2

During NBS Visit 2 (or NBS Visit 4), one fasting blood draw is taken and participants provide two spot urine

collections for the DLW procedures. The participants bring in their 24-hour urine collection for processing.

Processing and shipping samples occurs as follows:

CC staff process and freeze samples from the blood and urine specimens in preparation for shipping.

CC staff ship specified serum, plasma, and urine samples to McKesson BioServices in Rockville, MD. McKesson

BioServices ships the samples to the designated labs for analysis.

Laboratory quality control occurs as follows:

Five percent of biospecimen samples will have blinded duplicate aliquots collected for laboratory quality control

(QC). Two CCs (Pittsburgh and Stanford) will prepare all of the NBS QC samples.

NBS Manual Page 40


7.1 General Guidelines

7.1.1 Safety Procedures - Precautions for Handling Blood

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.1.1 - Safety Procedures - Precautions for

Handling Blood).

7.1.2 Safety Procedures - Precautions for Handling Urine

Follow relevant safety precautions described in WHI Manuals: Volume 2 – Procedures; Section 11.1.1 -

Safety Procedures - Precautions for Handling Blood. Additionally, follow institutional, local, and state

guidelines and requirements for safe handling of urine as a biological specimen.

7.1.3 Training and Certification

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.1.2 – Training and Certification.

NBS specifics:

A WHI CC staff person may draw, process and ship the blood and urine specimens for the NBS after

completing the NBS training activities and being certified for specified WHI tasks (refer to NBS

Manual Section 2.2.2 – Non-Lead NBS Staff).

7.1.4 Facilities

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.1.3 –Facilities.

7.1.5 Supplies

Refer to NBS Manual, Section 2.4.4 – Guidelines for Ordering Supplies for information about supplies for

blood and urine collection, processing and shipment.

7.1.6 Labels

The CCC provides the CCs with NBS label sets. There is one NBS label set per participant. This means

that all the blood and urine specimens collected from a participant at NBS Visit 1 and NBS Visit 2 have the

same specimen identification number. Each label set contains 42 labels, all of which are needed for the NBS

Visit 1 and NBS Visit 2 forms, sample tubes, and sample aliquots. Refer to Figure 7.1 NBS Label Set (Visits

1 and 2).

There are 18 labels for NBS Visit 1: 10 labels with bar codes to be used for the form, DLW spillage,

blood tube, blood and urine aliquots, plus 8 labels without bar codes used as descriptors or blank

labels.

There are 24 labels for NBS Visit 2: 19 labels with bar codes to be used for the form, indirect

calorimetry (for CCs conducting indirect calorimetry), blood tube, blood and urine aliquots, plus

5 labels without bar codes used as descriptors or blank labels.

NBS Visits 3 and 4 have a similar label set and are labeled appropriately with Visit 3 and Visit 4 text.

Place the participant’s NBS label set in her chart. Write the participant’s name on one of the blank labels in

the NBS label set or place the participant’s WHI member ID label on the back of the NBS label set so that it

can easily be identified as belonging to that specific participant.

NBS Manual Page 41


KEY POINTS (about the NBS label set):

Each label set has an identification number.

Label all the specimens collected at NBS Visits 1 and 2 with this identification number using the

appropriate bar-coded NBS label.

Label Form 75 – NBS Visit 1 and Form 76 – NBS Visit 2 with this identification number using the

appropriate bar-coded NBS label. There is only one label for each of the NBS Visit forms. During data

entry, there will be a check on key-entry of Form 76 – NBS Visit 2 to ensure that that the label set

identification number matches the one entered on Form 75 – NBS Visit 1.

When time points are involved in specimen collection, those times are explicitly displayed on the label

and the last digit of the aliquot matches the time point. For example, the NBS Visit 1 spot urine aliquots

are as follows: 30=Time 0, 32=Time 2, 33=Time 3, 34=Time 4.

The 2-digit aliquot identification numbers remain consistent across label sets for different participants.

Only the label set identification number differs between participants.

The same key points about the NBS label set apply to NBS Visits 3 and 4.

Figure 7.1 – NBS Label Set (Visits 1 and 2) provides a sample label set for NBS Visits 1 and 2.

NBS Manual Page 42


Figure 7. 1 NBS Label Set (Visits 1 and 2)

**********

NBS

Label Set

920003 M

**********

NBS

VISIT 1

DO NOT USE

[BAR CODE]

920003 M

FORM 75

NBS

Visit 1

[BAR CODE]

920003 M

NBS DLW

SPILLAGE

Container

Visit 1

NBS

Visit 1

Spot Urine

DO NOT USE

[BAR CODE]

920003-30

NBS URINE

Aliquot

Visit 1

Time 0

[BAR CODE]

920003-32

NBS URINE

Aliquot

Visit 1

Time 2

[BAR CODE]

920003-33

NBS URINE

Aliquot

Visit 1

Time 3

[BAR CODE]

920003-34

NBS URINE

Aliquot

Visit 1

Time 4

NBS

Visit 1

60+ Blood

DO NOT USE

[BAR CODE]

920003 M

NBS BLOOD

Tube

Visit 1

[BAR CODE]

920003-40

Lavender

Aliquot NBS

Plasma

Visit 1

[BAR CODE]

920003-41

Lavender

Aliquot NBS

Plasma

Visit 1

[BAR CODE]

920003-42

Lavender

Aliquot NBS

Plasma

Visit 1

NBS Manual Page 43


Figure 7.1 NBS Label Set (Visit 1 and 2) continued

NBS

VISIT 2

DO NOT USE

[BAR CODE]

920003 M

FORM 76

NBS

Visit 2

[BAR CODE]

920003 M

NBS

Indirect

Calori-

metry

NBS

Visit 2

Spot Urine

DO NOT USE

[BAR CODE]

920003-50

NBS URINE

Aliquot

Visit 2

Time 0

[BAR CODE]

920003-51

NBS URINE

Aliquot

Visit 2

Time 1

NBS

Visit 2

Fasting Blood

DO NOT USE

[BAR CODE]

920003 M

NBS BLOOD

Tube

Visit 2

[BAR CODE]

920003 M

NBS BLOOD

Tube

Visit 2

[BAR CODE]

920003 M

NBS BLOOD

Tube

Visit 2

[BAR CODE]

920003 M

NBS BLOOD

Tube

Visit 2

[BAR CODE]

920003-02

Royal Blue

Aliquot NBS

Serum

Visit 2

[BAR CODE]

920003-03

Royal Blue

Aliquot NBS

Serum

Visit 2

[BAR CODE]

920003-04

Royal Blue

Aliquot NBS

Serum

Visit 2

[BAR CODE]

920003-05

Royal Blue

Aliquot NBS

Serum

Visit 2

[BAR CODE]

920003-10

Lavender

Aliquot NBS

Plasma

Visit 2

[BAR CODE]

920003-11

Lavender

Aliquot NBS

Plasma

Visit 2

[BAR CODE]

920003-12

Lavender

Aliquot NBS

Plasma

Visit 2

NBS

Visit 2

24-hour

Urine

DO NOT USE

[BAR CODE]

920003 M

NBS URINE

Tube

Visit 2

24-hour

[BAR CODE]

920003-60

NBS URINE

Aliquot

Visit 2

24-hour

[BAR CODE]

920003-61

NBS URINE

Aliquot

Visit 2

24-hour

[BAR CODE]

920003-62

NBS URINE

Aliquot

Visit 2

24-hour

NBS Manual Page 44


7.1.7 Quality Control (QC) for Laboratory Analysis

Laboratory quality control will be assessed on a 5% subsample of NBS specimens, i.e., specimens from 30

participants NBS-wide for NBS Visits 1 and 2 and from 6 participants NBS-wide for NBS Visits 3 and 4.

The analytic laboratories will receive the QC samples as blinded duplicate aliquots.

For efficiency, two CCs will prepare the NBS QC samples. Each of the two QC CCs will prepare samples

from 15 participants at NBS Visits 1 and 2 and from 3 participants at NBS Visits 3 and 4. The QC

participants need to remain the same for both NBS Visits (i.e., participants identified for QC at NBS Visit 1

will continue to be part of the QC at NBS Visit 2).

Blood QC.

3-hour post-DLW plasma (for women 60 years of age or older). For NBS Visit 1 (or NBS Visit 3),

two of the three 3-hour post-DLW plasma aliquots will be labeled for QC.

Fasting serum and plasma. For NBS Visit 2 (or NBS Visit 4), fasting serum and plasma samples

collected from the WHI banked QC blood repository will be used for NBS QC. Staff do not need to

prepare NBS QC serum or plasma aliquots for NBS Visit 2 (or NBS Visit 4).

Participants will not need to have additional blood drawn for NBS QC at any of the NBS Visits.

Urine QC.

DLW Spot Urine Collection. For each DLW spot urine collection at all NBS Visits, staff will

prepare and label two additional 4 mL aliquots for QC.

24-hour Urine Collection. For the 24-hour urine collection processed at NBS Visit 2 (or NBS Visit

4), staff will prepare and label six additional 1.8 mL aliquots for QC.

Participants will not need to provide additional urine collections for NBS QC for either the DLW

spot urine collections or the 24-hour urine collection.

For QC details related to each NBS Visit, refer to NBS Manual Section 7.2.1.2. Quality Control (QC)

Samples for NBS Visit 1 (or NBS Visit 3) and NBS Manual Section 7.3.1.2. Quality Control (QC) Samples for

NBS Visit 2 (or NBS Visit 4) - QC CCs Only.

NBS Manual Page 45


7.2 NBS Visit 1 (or NBS Visit 3)

7.2.1 General Blood and Urine Sampling and Aliquot Schedule and Quality Control (QC)

7.2.1.1 Sampling and Aliquot Schedule

TIME SAMPLE ALIQUOT QC

(for the two QC CCs)

Time 0

(Fasting)

Spot urine One, 4 mL

(5 mL cryovial)

Two, 4 mL

(5 mL urine cryovial)

Time 2

(Two hours post-DLW)


(5 mL cryovial)

Two, 4 mL


Time 3

(Three hours post-DLW),


(5 mL cryovial)

Two, 4 mL


Time 3

(Three hours post-DLW)

Blood Draw

Lavender dry EDTA,

one 10 mL;

for participants 60 years of

age or older

Three 1.8 mL plasma

(2 mL cryovial)

Label two of the three

plasma aliquots with labels

set aside for QC.

Time 4

(Four hours post-DLW)


(5 mL cryovial)

Two, 4 mL


7.2.1.2 Quality Control (QC) Samples for NBS Visit 1 (or NBS Visit 3) - QC CCs only

Refer to NBS Manual, Section 7.1.7 Quality Control (QC) for Laboratory Analysis for general blood and

urine QC information.

The two QC CCs will collect QC aliquots by convenience sampling until the specified number of participants

have had QC specimens collected. For a NBS QC participant at NBS Visit 1 (or NBS Visit 3), all urine QC

aliquots must be from the same participant, and the same participant must have three plasma aliquots.

For NBS Visit 1, each of the two QC CCs will collect QC aliquots from 15 participants (for a total of

30 participant-sets NBS-wide).


6 participant-sets NBS-wide).

Labels. For each QC participant, use two additional NBS participant label sets for the NBS Visit forms and

QC aliquots.

Blood. For each QC participant 60 years of age or older, label two of the three plasma aliquots at NBS Visit

1 (or NBS Visit 3) with labels reserved for QC.

Urine. For the spot urine collections at NBS Visit 1 (or NBS Visit 3), collect two additional 4 mL aliquots

per NBS QC participant at each time point. Label one of these additional aliquots from one set of the two

participant label sets reserved for QC and label the second additional aliquot from the remaining participant

label set reserved for QC.

NBS Manual Page 46


7.2.2 Day of NBS Visit 1 (or NBS Visit 3) (Day 1)

7.2.2.1 Before the Participant Arrives

1. Obtain a Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3).

2. Obtain one NBS label set for the participant. (For QC CCs, obtain 3 label sets for each QC participant.)

3. Insert the Form 75 – NBS Visit 1 and the NBS label set into the participant’s file (or Form 77 – NBS Visit

3 for the reliability subsample). Write the participant’s name on one of the blank labels in the NBS label

set or place the participant’s WHI member ID label on the back of the NBS label set so that it can easily

be identified as belonging to that specific participant.

4. Arrange the lavender dry EDTA tube in a test tube rack (for participants 60 years of age and older).

Arrange corresponding 2 mL cryovials in rack.

5. Arrange the 5 mL cryovials in a rack for the spot urine aliquots.

7.2.2.2 During the Visit (Overview)

1. Verify that the participant is eligible and has signed the NBS Consent by reviewing Form 75 – NBS Visit

1 (or Form 77 – NBS Visit 3), question numbers 5, 6, and 7.

2. Apply the NBS form label with the identification number on Form 75 – NBS Visit 1 (or Form 77 – NBS

Visit 3). For participants in the QC subsample, label the appropriate section of the NBS form.

3. Review with the participant the blood and urine collection procedures for NBS Visit 1 (or NBS Visit 3).

4. Collect the spot urine samples for Time 0 (NBS Manual Section 7.2.2.2.3), Time 2 (NBS Manual, Section

7.2.2.2.4), and Time 3 (NBS Manual, Section 7.2.2.2.5).

5. For participants 60 years of age or older, draw a blood sample 3 hours after DLW ingestion, in

conjunction with the 3-hour spot urine collection (NBS Manual, 7.2.2.2.6).

6. Collect the spot urine sample for Time 4 (NBS Manual, Section 7.2.2.2.7).

7. Instruct the participant on the 24-hour urine collection and give her the 24-hour urine collection kit (NBS

Manual, Section 7.2.2.2.8).

7.2.2.2.1 Timing of the Spot Urine Collections

The timing for each of the spot urine collections during the visit do not need to be precisely “on the hour,” but

the exact times do need to be recorded on Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3). Subsequent

collections may be collected at their scheduled times.

Example 1: NBS Visit 1. A woman ingests the DLW at 9:00 AM. She is unable to produce a urine

sample 2 hours after ingestion of the DLW (11:00 AM), but is able to 2-1/2 hours after (11:30 AM).

On Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3), note 11:30 AM for the Time 2 Urine

Collection. Obtain the Time 3 Urine Collection at 12:00 PM as scheduled.

Example 2: NBS Visit 1. A woman ingests the DLW at 9:00 AM and she needs to void 90 minutes

later (10:30 AM). On Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3), note 10:30 AM for the Time

2 Urine Collection. Obtain the Time 3 Urine Collection at 12:00 PM as scheduled.

Coordinate the timing of the 3-hour urine and blood collections for women 60 years of age and older to occur

together, i.e., if the urine collection needs be earlier or later than the scheduled Time 3 collection, collect the

blood draw immediately after the urine collection.

NBS Manual Page 47


7.2.2.2.2 DLW Spot Urine Collection Steps

At each time point when collecting the spot urine samples for the doubly labeled water analysis, follow these

six steps:

1. Place the urine collection hat on the toilet (under the toilet seat) and ask participant to void into the

hat. If she needs to have a bowel movement, she should remove the hat, have the bowel movement,

and then replace the hat for the urine collection.

2. Complete the appropriate section of the NBS Visit forms.

3. Attach appropriate labels to cryovials. For participants in the QC subsample, label the extra

cryovials.

4. Using a disposable transfer pipette, fill the labeled 5 mL Corning cryovial with 4 mL of urine

specimen and store in freezer. Do not fill the cryovial to the top in order to allow for volume

expansion upon freezing. For participants in the QC subsample, fill two additional labeled 5 mL

Corning cryovials, each with 4 mL of urine specimen. Urine samples for the DLW analyses do not

need to be centrifuged.

5. Discard remaining urine by flushing down the toilet.

6. Rinse and dry hat. It is critical that the hat by thoroughly dried.

Refer to NBS Manual, Section 5.1.4.10.1 – Handling Collection Problems for guidelines on handling

problems with delayed or insufficient urine collection.


Before starting the visit procedures, verify that the participant is eligible and has signed the NBS Consent by

reviewing Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3), question numbers 5, 6, and 7.

Collect the fasting spot urine sample by following steps in the NBS Manual, Section 7.2.2.2.2 - DLW Spot

Urine Collection Steps. Note the exact collection time on Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3).

7.2.2.2.4 Time 2 Urine Collection (2 hours after DLW ingestion)

Collect the 2-hour spot urine sample by following steps in the NBS Manual, Section 7.2.2.2.2 - DLW Spot

Urine Collection Steps. The time does not need to be exactly at two hours; however, note the exact collection

time on Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3).



Urine Collection Steps. The time does not need to be exactly at three hours; however, note the exact

collection time on Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3).

NBS Manual Page 48


7.2.2.2.6 Time 3 Blood Draw (3 hours after DLW ingestion, for women 60 years of age or older)

Collect the 3-hour blood draw for women 60 years of age or older, noting the exact time on Form 75 – NBS

Visit 1 (or Form 77 – NBS Visit 3). Coordinate the time to occur just after the 3-hour spot urine

collection. Note the exact time on the NBS Visit form.

1. Follow general preparation for the participant (refer to NBS Manual, Section 7.4.2. Preparation for

the Participants)

2. Ensure the participant understands that you will draw 2 teaspoons of blood from her arm.

3. Following WHI venipuncture procedures (NBS Manual, Section 7.4.3. Venipuncture), draw a single

10 mL lavender-dry EDTA tube 3 hours after the DLW ingestion. Attempt to draw a full tube. If

the venipuncture is not successful or if you do not draw enough sample on the first occasion to fill

the tube at least halfway, ask the participant for permission to attempt a blood draw a second time.

If on the second attempt, less than 10 mL is collected, process the total amount collected. Refer to

NBS Manual Section 7.4.4.7 Blood and Urine Collection, Processing, and Shipping; Deficient

Serum or Plasma Samples. Note: If the 3-hour spot urine collection is slightly delayed, then delay

the 3-hour blood draw to immediately after that urine collection.

4. Note any missing collections in the “Notes” section of Form 75 - NBS Visit 1. Follow the

procedures outlined in the NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify the CCC.

5. Check the identifying information on the form and the labels to make sure the form is correctly

labeled.

The 3-hour post-DLW blood draw sample does not need to be protected from light.

Bring the lavender tube to the blood processing area. Process the blood according to NBS Manual,

Section 7.5 - Blood Processing.



Urine Collection Steps. The time does not need to be exactly at four hours; however, note the exact

collection time on Form 75 – NBS Visit 1 (or Form 77 – NBS Visit 3).

7.2.2.2.8 Instructions for the 24-hour Urine Collection

For participants who receive PABA (B-vitamin) tablets:

Add boric acid powder to the urine collection bottles (boric acid supplied by the CCC).

Before giving the urine collection bottles to participants, add 2.0 gram of boric acid powder to

each urine collection bottle. Measure the boric acid powder using a gram scale. If a gram scale is

not available, add 1.5 level measuring teaspoons of boric acid powder to each bottle*. Use

standard safe laboratory practices by wearing a mask and gloves when adding the boric acid

powder to the urine collection bottles. Boric acid is a weak base, although it can be slightly

irritating if inhaled.

* The boric acid may be weighed or measured using household measuring spoons labeled, “for

boric acid use only.” When handling the boric acid, wear disposable gloves and preferably a

disposable mask. Even though boric acid is a safe preservative, take precautions to avoid skin

contact or inhalation.

Place two stickers on each urine collection bottle: one sticker that reads “Leave powder preservative

inside bottle” and a second sticker that reads “Remember…take the PABA (B-vitamin) tablets”

(stickers supplied by the CCC).

NBS Manual Page 49


General instructions for the 24-hour urine collection

Refer to NBS Manual, Section 5.1.4.16 – Explain and Provide Materials for 24-hour Urine Collection

for instructing participants how to conduct the 24-hour urine collection.

NBS Manual Page 50


7.3 NBS Visit 2 (or NBS Visit 4) (Day 15)

7.3.1 General Blood and Urine Sampling and Aliquot Schedule and Quality Control (NBS-modified)

7.3.1.1 Sampling and Aliquot Schedule

TIME SAMPLE ALIQUOT QC

(for the two QC CCs)

Time 0

(Fasting)


(5 mL cryovial)

Two, 4 mL

(5 mL cryovial)

Time 0

(Fasting)

Blood

Royal Blue, three 7 mL

COLLECT BEFORE

LAVENDER DRY EDTA

Four 1.8 mL Serum

(2 mL cryovial)

No NBS aliquots needed for

QC. QC samples will be

pulled from WHI banked

QC samples.

Time 0

(Fasting)

Blood

Lavender (dry EDTA),

one 10 mL

COLLECT AFTER

ROYAL BLUE SERUM

Three 1.8 mL Plasma

(2 mL cryovial)

No NBS aliquots needed for

QC. QC samples will be

pulled from WHI banked

QC samples.

Received from participant 24-hour urine collection Three, 1.8 mL

(2 mL cryovial)

Six, 1.8 mL

(2 mL cryovial)

Time 1

(One hour after Time 0)


(5 mL cryovial)

Two, 4 mL

(5 mL cryovial)

7.3.1.2 Quality Control (QC) Samples for NBS Visit 2 (or NBS Visit 4) - QC CCs only

Refer to NBS Manual, Section 7.1.7 - Quality Control(QC) for Laboratory Analysis for general blood and

urine QC information.

The two QC CCs will collect QC aliquots by convenience sampling until the specified number of participants

have had QC specimens collected. For an NBS QC participant at NBS Visit 2 (or NBS Visit 4), all urine QC

aliquots must be from the same participant, and the same participant must have sufficient plasma for one

sample aliquot and two QC aliquots.


30 participants NBS-wide).


6 participants NBS-wide).

Labels. For each QC participant, use two additional NBS participant label sets for the NBS Visit forms and

QC aliquots. For NBS Visit 2 (or NBS Visit 4) carry forward use of the label sets stored with the participant

chart from NBS Visit 1 (or NBS Visit 3).

Blood. Staff do not need to collect NBS QC serum or plasma aliquots at NBS Visit 2 (or NBS Visit 4).

Quality control of blood sample analyses for NBS Visit 2 will be done using 30 blind duplicate pairs from

banked WHI QC samples. For the 20% reliability subsample at NBS Visit 4, two pairs of banked WHI blind

duplicate samples will be analyzed for QC.

NBS Manual Page 51


Urine:

For spot urine collections at NBS Visit 2 (or NBS Visit 4), collect two additional 4 mL aliquots per

NBS QC participant at each time point. Label one of these additional aliquots from one set of the

two participant label sets reserved for QC and label the second additional aliquot from the remaining

participant label set reserved for QC.

For the 24-hour urine collection, collect six additional 1.8 mL aliquots for each NBS QC participant.

Label three of these additional aliquots from one set of the two participant label sets reserved for QC

and label the second three additional aliquots from the remaining participant label set put aside.

7.3.2 One Day Before NBS Visit 2 (or NBS Visit 4) (Day 14)

Refer to NBS Manual, Section 5.2.2.2 - Pre-Visit 2 Reminder Call and Section 5.222.1 – Review Instructions

for 24-hour Urine Collection (Day 14)

7.3.3 Day of NBS Visit 2 (or NBS Visit 4) (Day 15)

7.3.3.1 Before the Participant Arrives

1. Obtain Form 76 – NBS Visit 2 (or Form 78 – NBS Visit 4).

2. Apply the NBS form label with the identification number on the NBS Visit 2 form.

3. Insert the labeled Form 76 – NBS Visit 2 and the label set into the participant’s file (or Form 78 –

NBS Visit 4).

4. Arrange the three blue serum tubes and one dry lavender EDTA tubes in a test tube rack.

5. Arrange the two 5 mL urine cryovial tubes in a rack.

7.3.3.2 During the Visit (Overview)

1. Verify that the NBS Visit 2 Participant Update Worksheet has been completed and that before the

blood draw is taken that the participant has been fasting for six hours.

2. Review with the participant the blood and urine visit procedures for NBS Visit 2 (or NBS Visit 4).

3. Receive the 24-hour urine collection from participant (NBS Manual, Section 7.3.3.2.1).

4. Collect the Time 0 (fasting) spot urine sample (NBS Manual, Section 7.3.3.2.2).

5. Draw the Time 0 (fasting) blood sample (NBS Manual, Section 7.3.3.2.3).

6. Collect the Time 1 (1-hour) spot urine sample (NBS Manual, Section 7.3.3.2.4).

7. Process the 24-hour urine collection (NBS Manual, Section 7.3.3.2.5).

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7.3.3.2.1 Receive the 24-hour Urine Collection From Participant

1. Mark the participant’s name on the collection container(s) using a Sharpie pen or write her name

on a blank label from the participant’s label set and place the collection container(s) in the

refrigerator until after the Time 0 (Fasting) Urine Collection and Time 0 (Fasting) Blood Draw have

been completed. Refer to NBS Manual, Section 7.3.3.2.5 - 24-hour Urine Collection Processing for

the specific steps for processing the 24-hour urine collection.

2. Attach a WHI participant member ID label to Record Sheet for 24-hour Urine Collection (last page -

Instructions for 24-hour Urine Collection, given at NBS Visit 1).

3. Review the Record Sheet for 24-hour Urine Collection with the participant to ensure all pertinent

questions have been answered. The goal is to gather information about missed or contaminated

urine collections.

4. The following responses on the participant’s Record Sheet for 24-hour Urine Collection indicate the

potential of a missed, spilled, or contaminated 24-hour urine collection:

‘Yes’ response to Qx. 1: “Did you miss any urine collections?”

‘No’ response to Qx. 4: “If you were away from home, were you able to collect your urine?”

‘No’ response to Qx. 5: “Were you able to collect your urine during the night?”

‘Yes’ response to Qx. 6: “Did you have any diarrhea during the collection period.”

‘Yes’ response to Qx. 7: “Did you spill any urine when pouring it into the bottle.”

5. If the participant indicates that she missed any urine collections, use Qx. 2 – “If you missed urine

collections, how many did you miss?” on the Record Sheet for 24-hour Urine Collection to note the

total number of collections the participant missed.

6. For participants who have indicated a missed, spilled, or contaminated 24-hour urine collection,

follow the procedures described in the NBS Manual, Section 8.2.1 - Notes on NBS Forms to notify

the CCC.

7. Store the Record Sheet for 24-hour Urine Collection in the participant’s file.

8. Use the NBS Visit 2 (or Visit 4) PABA Worksheet for Staff to record the following information about

the PABA (B-vitamin) tablets:

a. Qx. 1: Did the 24-hour urine kit contain the 3 PABA (B-vitamin) tablets? (‘Yes’ or ‘No’). This

question should have been marked by staff at the participant’s NBS Visit 1 (or Visit 3). If not,

verify whether or not the participant’s urine kit contained the 3 PABA (B-vitamin) tablets and

mark the appropriate response.

b. Qx. 2 (only if ‘Yes’ to Qx. 1): How many PABA (B-vitamin) tablets did the participant take?

If the participant does not have the packaging with her, ask her how many of the PABA (B-

vitamin) tablets that she took (‘0’, ‘1’, ‘2’, ‘3’, or ‘Don’t know’).

c. Qx. 3 (only if ‘Yes’ to Qx. 1): Did the participant take any acetaminophen (for example

Tylenol®) during the urine collection? (‘Yes’, ‘No’ or ‘Don’t know’).

d. Qx. 4 (only if ‘Yes’ to Qx. 1): Did the participant take any vitamins, other than the PABA (B-

vitamin) tablets during the urine collection? (‘Yes’, ‘No’ or ‘Don’t know’).

9. Staple the NBS Visit 2 (or Visit 4) PABA Worksheet for Staff to Form 76 – NBS Visit 2 (or Form 78 –

NBS Visit 4).


Collect the fasting spot urine sample following the steps outlined in NBS Manual, Section 7.2.2.2.2 –

DLW Spot Urine Collection Steps. Note the exact time on Form 76 – NBS Visit 2 (or Form 78 – NBS

Visit 4).

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7.3.3.2.3 Time 0 (Fasting) Blood Draw

Collect the fasting blood draw.

1. Verify that the participant has been fasting for six hours.

2. Follow general preparation for the participant (refer to NBS Manual, Section 7.4.2. Preparation for

the Participants).

3. Ensure the participant understands that you will draw 2 Tablespoons of blood from her arm.

4. Follow the WHI venipuncture procedures (refer to NBS Manual, Section 7.4.3. Venipuncture).

Draw the royal blue tubes first, followed by the lavender tube.

The reason that the royal blue tubes are drawn first is because they are preservative-free for

trace element analysis. By drawing them first, the risk is lessened for contamination from the

lavender EDTA tube.

Attempt to draw the full set of blood collection tubes. If you do not draw enough sample on the

first occasion, follow the procedures in NBS Manual, Section, 7.4.4.7 Deficient Serum and

Plasma Samples. The remainder of NBS Visit 2 may continue without the blood draw.

All of these samples must be protected from heat and light. Prepare aluminum sleeves for all of

the tubes. Samples from these tubes will be analyzed for carotenoids, folate, and other nutrients

which break down in white light. The aluminum foil cover will help protect the blood from

light and prevent deterioration of the carotenoids.

5. Check the identifying information on the form and the labels to make sure that all are correctly

labeled.

7.3.3.2.4 Time 1 Urine Collection (1 hour after Time 0 [fasting] urine collection)

Collect the 1-hour spot urine sample following the steps outlined in NBS Manual, Section 7.2.2.2.2 – DLW

Spot Urine Collection Steps The time does not need to be exactly at one hour; however, note the exact time

on Form 76 – NBS Visit 2 (or Form 78 – NBS Visit 4).

7.3.3.2.5 24-hour Urine Collection Processing

The 24-hour urine collection may be processed between the Time 0 and Time 1 Urine Collections if time is

available, or after the two spot urine collections are completed. Follow these steps for processing the 24-hour

urine collection:

1. Measure the total volume of urine collected using the graduations on the side of the urine collection

container. Estimate to the nearest 25 mL graduation line, which is the smallest graduation on the

bottle. If needed, use an appropriate graduated cylinder.

2. Document the total volume on Form 76 – NBS Visit, Qx. 11.2 (or Form 78 – NBS Visit 4, for the

reliability subsample). Staff do not need to inspect the urine for color or degree of cloudiness. The

lab will note any questionable samples.

3. Mix the 24-hour urine collection in its container thoroughly. If the participant brings in more than

one 3-liter container, mix the contents of the containers together using the steps outlined below:

a. Gently swirl the urine in each collection bottle.

b. Then:

Option 1: If you have access to a large (e.g., 4 L) flask, beaker, or graduated cylinder,

combine the urine from the two collections. Mix gently.

Option 2: If you do not have access to a large (e.g., 4 L) flask, beaker, or graduated

cylinder:

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Gently pour contents from one collection bottle to the other 3 three times,

swirling the collections each time to enhance mixing. Even though the entire

collection will not be in a single container, the repeated mixing of portions of

the collections should allow adequate mixing.

EXAMPLE: a participant brings in 4 L of urine, 3 L in one collection bottle (Bottle A) and 1 L in

the other bottle (Bottle B).

a. Measure and record the volumes in each collection bottle.

b. Gently swirl the contents of each bottle to mix the respective samples.

c. Carefully pour 2 L from the Bottle A into Bottle B and gently swirl Bottle B.

d. Carefully pour 2 L from Bottle B into Bottle A and gently swirl Bottle A.

e. Carefully pour 2 L from Bottle A into Bottle B and gently swirl Bottle B.

4. Centrifuge 8 mL of the well mixed urine collection, using the 10 mL tubes provided for urine

centrifugation. For QC CCs, centrifuge two additional 8 mL urine samples. Process the 24-hour

urine for 5 minutes at 1,300 xg (per WHI Manuals, Vol. 2 – Procedures; Section 11.6 Urine

Processing).

The intent of 24-hour urine centrifugation is to exclude particulates, but a small amount of

sediment in the sample is acceptable.

5. Attach the appropriate labels to each cryovial.

6. Using a disposable transfer pipette, aliquot the urine by placing:

1.8 mL of urine into each of three labeled 2 mL cryovials.

For QC CCs, 1.8 mL of urine into each of six additional labeled 2 mL cryovials (three

cryovials for each QC set).

Be careful not to overfill the cryovials because urine volume will increase upon freezing

and the cryovials may crack.

7. Place aliquots in the -70 degree Centigrade freezer.

7.4 Blood Draw Specifics

7.4.1 Preparation of the Blood Drawing and Processing Areas for All NBS Visits

Ensure the blood drawing and blood processing areas are equipped with the proper supplies.

7.4.2 Preparation for the Participants

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.4.5 – Participant Preparation.

NBS specifics:

Each participant must have signed the NBS Consent authorizing the CC to draw blood before you

can draw her blood.

Ensure the participant understands the general aspects of blood collection as detailed in the NBS

Consent and WHI informed consent.

Verify with the participant that she has been fasting for 6 hours at NBS Visit 2. If needed, refer to

NBS Manual, Section 7.4.4.1 – Directions for Staff When Participants Not Fasted.

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7.4.3 Venipuncture

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.5 – Venipuncture.

NBS specifics:

Refer to NBS Manual, Section 7.2 – NBS Visit 1 (or NBS Visit 3) and NBS Manual, Section 7.3 –

NBS Visit 2 (or NBS Visit 4) for the NBS sampling and aliquot schedule.

Complete the remaining relevant sections of Form 75 – NBS Visit 1 (for NBS Visit 1), Form 76 –

NBS Visit 2 (for NBS Visit 2), Form 77 – NBS Visit 3 (for NBS Visit 3), or Form 78 – NBS Visit 4

(for NBS Visit 4). Note a missing blood draw in the “Notes” section of the NBS Visit form.

Follow the procedures outlined in the NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify

the CCC.

Deliver the blood sample tube(s) and Form 75 – NBS Visit 1 (for NBS Visit 1) or Form 76 – NBS

Visit 2 (for NBS Visit 2) to the blood processing area. Do the same for NBS Visits 3 and 4 for the

reliability subsample, using Form 77 – NBS Visit 3 or Form 78 – NBS Visit 4, respectively.

7.4.4 Blood Collection Problems

7.4.4.1 Directions for Staff When Participants Are Not Fasted

For NBS Visit 2, do not draw the blood sample if the participant has not been fasting for at least 6 hours

(water, black coffee or tea is OK). Note: A participant is considered to be non-fasting if she has consumed

food, or caloric beverages within the 6 hours before the blood draw.

If she is not fasting and is:

Willing to wait at the CC until the required number of hours of fasting has occurred, proceed with

the blood draw at that time.

Not willing to wait at the CC until the required number of hours of fasting has occurred, ask her if

she is willing to reschedule her appointment.

If she is:

Willing to reschedule - reschedule an appointment for the fasting blood draw, and then proceed

with the remaining NBS Visit 2 tasks. Complete the appropriate section of the NBS Visit 2

Participant Update Worksheet.

Not willing to reschedule - proceed with the remaining NBS Visit 2 tasks. Complete the

appropriate section of the NBS Visit 2 Participant Update Worksheet and follow the procedures

outlined in the NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify the CCC.

7.4.4.2 Handling Participants Who Are Extremely Apprehensive About Having Blood Drawn

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.4.6 - Handling Participants Who Are Extremely

Apprehensive About Having Blood Drawn.

7.4.4.3 Special Consideration for Drawing Blood in the Elderly

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.6.1 - Special Consideration for Drawing Blood

in the Elderly.

7.4.4.4 Difficult-to-Identify Venipuncture Sites

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.6.2 – Difficult-to-Identify Venipuncture Sites.

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7.4.4.5 Difficult Venipuncture

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.6.3 – Difficult Venipuncture.

NBS specifics:

The participants may be dehydrated having fasted for 6 hours (except water or black coffee or tea) in

preparation for NBS Visit 2. Encourage the participants to drink water liberally during the fast. If

this is a particular problem at your CC, consider offering the participants water as they arrive at the

visit.

If venipuncture for NBS Visit 1 (or NBS Visit 3) fails with the second attempt or second technician,

do not attempt again. Proceed with the remaining tasks for NBS Visit 1 (or NBS Visit 3). Note the

missing blood draw in the “Notes” section of the NBS Visit form. Follow the procedures

outlined in the NBS Manual, Section 8.2.1 – Notes on NBS Forms to notify the CCC.

If venipuncture for NBS Visit 2 (or NBS Visit 4) fails with the second attempt or second technician,

ask the participant if she is willing to return at another time for a venipuncture. (Refer to NBS

Manual, Section 7.4.4.7 - Deficient Serum and Plasma Samples). Then proceed with the remaining

tasks for NBS Visit 2 (or NBS Visit 4).

7.4.4.6 Fainting

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.2.6.4 – Fainting.

7.4.4.7 Deficient Serum and Plasma Samples

Deficient blood from venipuncture:

In general: If the venipuncture is not successful (no blood obtained) ask the participant for

permission to attempt a second blood draw. Do not attempt a third time.

Blue tube: If you do not get a full royal blue-stoppered vacutainer of blood (for serum), you should

try to collect an additional half-tube immediately, with permission. After centrifuging the tubes, you

can combine the serum from the half-tube with the serum from the first incompletely filled royal

blue-stoppered tubes.

Lavender tube: If you do not draw enough sample on the first occasion to fill the lavender tube at

least halfway, attempt a blood draw a second time, with permission. If you have two partially filled

lavender tubes, centrifuge each one and aliquot the plasma per procedures described in the NBS

Manual, Section 7.5.3.1 Preparation of Cryovials.

Deficient plasma or serum after centrifugation:

Do not draw additional blood if plasma or serum is inadequate after centrifuging the blood samples.

7.5 Blood Processing

7.5.1 Blood Sample Handling and Processing: Timelimits

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.3.1 – Timelimits and Sample Handling.

NBS specifics:

The only Vacutainers being used for NBS are the lavender dry EDTA and royal blue.

Always protect all the blood samples collected at NBS Visit 2 (or NBS Visit 4) from natural and

fluorescent white light. Store samples in covered containers or tubes.

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7.5.2 Operating the Refrigerated Centrifuge

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.3.2 – Operating the Refrigerated Centrifuge.

7.5.3 Processing Blood Samples

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.3.3 – Processing Samples.

NBS specifics:

The only Vacutainers being used for NBS are the lavender dry EDTA and royal blue.

Always protect all the blood samples collected at NBS Visit 2 (or NBS Visit 4) from natural and

fluorescent white light. Store samples in covered containers or tubes.

Place the samples in a freezer box dedicated to NBS. Complete the relevant portions of Form 75 –

NBS Visit 1 (for NBS Visit 1), Form 76 – NBS Visit 2 (for NBS Visit 2) (and for the reliability

subsample; Form 77 – NBS Visit 3 [for NBS Visit 3], or Form 78 – NBS Visit 4 [for NBS Visit 4]).

7.5.3.1 Preparation of Blood Cryovials

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.3.3.1 – Preparation of Blood

Cryovials.

NBS specifics:

Inspect each centrifuged tube for hemolysis. Re-centrifuge any specimen that appears to contain red

cells suspended in the serum or plasma and then proceed with aliquotting, freezing and shipping.

The analytic laboratory (ies) will make note of samples that may not be usable.

Review Form 75 – NBS Visit 1 (for NBS Visit 1) or Form 76 – NBS Visit 2 (for NBS Visit Two) to

determine the number of cryovials to prepare. Do the same for NBS Visits 3 and 4 with respective

NBS Visit forms.

When transferring the serum and plasma to the cryovials:

Lavender-stoppered tube (NBS Visit 1 or NBS Visit 3; NBS Visit 2 or NBS Visit 4):

Remove the stopper from the Lavender-stoppered tube.

Transfer 1.8 mL of plasma into each of the three 2 mL cryovials. If you are not certain that you

have sufficient plasma to fill each of the three cryovials with 1.8 mL plasma, first fill each vial

with 1.0 mL plasma then add an additional 0.8 mL plasma to each vial until you run out of

plasma. Screw the lids onto the three cryovials. Place the three vials in a freezer box dedicated

to NBS and cover with a lid to protect the serum from white light. Insert the lavender stopper

back into the blood collection tube. Place the blood collection tube and the pipette tip in a

biohazard container.

Royal Blue stoppered tubes (NBS Visit 2 or NBS Visit 4):

Remove the stopper from the royal blue-stoppered tubes.

Transfer 1.8 mL of serum into each of the four 2 mL cryovials. If you are not certain that you

have sufficient serum to fill each of the four cryovials with 1.8 mL serum, first fill each of the

vials with 1.0 mL serum. Then add an additional 0.8 mL serum to each vial until you run out of

serum. This will help assure that you get sample in each of the four cryovials. Screw the lids

onto the four cryovials. Place the four vials in a freezer box dedicated to NBS and cover with a

lid to protect the serum from white light. Insert the royal blue stoppers back into the blood

collection tubes. Place the blood collection tubes and the pipette tip in a biohazard container.

Complete the relevant portions of the NBS Visit forms.

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7.5.4 Freezing and Storing Blood and Urine Cryovials

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.3.5 – Freezing and Storing Blood

Cryovials.

NBS specifics:

Store NBS aliquots in boxes dedicated to NBS. Do not mix NBS aliquots with WHI aliquots.

For the 2 mL cryovials: Use the same type of boxes as for WHI. Blood and urine aliquots may be in

the same box.

For the 5 mL DLW urine cryovials: Use the boxes ordered for NBS. Do not store 1.8 mL cryovials

in the boxes designated for the 5 mL aliquots.

Using a black Sharpie marker, mark NBS in large letters on the side of the top of the box and the

side of the bottom of each box.

Inventory stored aliquots in a way that facilitates shipping all samples from NBS Visit 1 and NBS

Visit 2 together for a given participant (and similarly for NBS Visit 3 and NBS Visit 4 for the

reliability subsample).

7.5.5 Equipment Quality Assurance

Refer to WHI Manuals: Volume 2 Procedures; Section 11.3.5.1 – Equipment Quality Assurance.

7.6 Blood Storage and Shipping

7.6.1 Shipping Schedule

In general, refer to WHI Manuals: Volume 2 – Procedures; Section 11.4.1 – Shipping Schedule.

NBS specifics:

Ship samples from NBS Visit 1 and NBS Visit 2 together for each participant; i.e., delay shipping a

participant’s Visit 1 samples until her NBS Visit 2 samples have been collected (and similarly for

NBS Visit 3 and NBS Visit 4).

Within the shipment box, a participant’s samples will be in different-sized small boxes (see NBS

Manual, Section 7.5.4 – Freezing and Storing Blood and Urine Cryovials):

The participant’s 2mL cryovials (for blood and 24-hour urine) will be in the same

type of boxes as used for WHI.

The participant’s 5mL cryovials (for spot urine) will be in the boxes ordered for NBS

(slightly taller than the boxes used for WHI samples).

Before sending the shipment, verify that all the specimens (blood, spot urines, and 24-hour

urine) have been collected and that all the NBS labels for the participant have the same

specimen ID.

Ship NBS samples in mid- June, August, and November of 2004; and in mid-February and end of

March in 2005 (or when all NBS Visits have been completed). Ship to McKesson BioServices on

this schedule regardless of the number of samples in the freezer.

CCs may ship both NBS and WHI boxes together in the same large shipment, but NBS specimens

need to be in separate small boxes, marked “NBS”.

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7.6.2 Mailing Instructions

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.4.1.1 – Mailing Instructions.

7.6.3 Packaging Instructions

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.4.2 – Packaging Instructions.

7.6.4 Receipt of Frozen Specimen Shipment at McKesson BioServices

Refer to WHI Manuals: Volume 2 – Procedures; Section 11.4.3 – Receipt of Frozen Specimen Shipment at

McKesson BioServices.

NBS Manual Page 58


SECTION 8

DATA MANAGEMENT

8.0 Introduction

This section provides information about NBS data management: NBS label sets, NBS data collection

forms, NBS data entry, and NBS WHILMA Upgrade Notes.

8.1 NBS Label Set

The CCC provides CCs with NBS label sets. These label sets are generic (any specimen ID set can be

given to a participant), but once a label set has been used for a participant it is critical that the same

specimen ID is used on all the participant’s forms and specimens for NBS Visit 1 and NBS Visit 2.

Each participant has one NBS label set that includes labels for NBS Visit 1 and NBS Visit 2. For

information about the NBS label sets, refer to NBS Manual, Section 7.1.6 – Labels.

8.2 NBS Data Collection Forms

For copies of NBS forms, NBS forms instructions, and NBS worksheets, refer to NBS Manual, Section 9

– NBS Forms and Worksheets.

8.2.1 Notes on NBS Forms

Use the procedures described below to document and handle notes/responses on NBS Visit forms,

worksheets and/or the participant’s Record Sheet for 24-hour Urine Collection.

8.2.1.1 Using the “Notes” Box’ on the NBS Forms

Record notes/responses on NBS Visit forms, worksheets and/or the participant’s Record Sheet for 24-

hour Urine Collection in the following situations:

There are missing tasks that prevent data entry of the NBS Visit forms (Form 75, Form 76, Form

77, or Form 78).

If a specific NBS task is missed or cannot be completed (i.e., woman refuses a blood draw or

is unable to provide a specific spot urine, even after having additional water), NBS staff

leave the appropriate question on the NBS form ‘blank’ and indicate the problem in the

“Notes” box at the end of the NBS Visit form.

There are out-of-sequence tasks that prevent data entry of the NBS Visit form (Form 75, Form 76,

Form 77, or Form 78).

The participant was not given PABA (B-vitamin) tablets because of past hypersensitivity (Form 75,

Form 77).

There are responses on NBS Visit 2 (or Visit 4) Participant Update Worksheet indicating potential

changes that could affect DLW results (questions 1-3).

There are responses on the Record Sheet for 24-hour Urine Collection indicating missing or

contaminated 24-hour urine collections (questions 1, 2, 4, 5, 6 and 7).

Refer to NBS Manual, Section 8.2.1.2 – Handling Notes on NBS Forms for information about handling

notes recorded on NBS forms.

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8.2.1.2 Handling Notes on NBS Forms

If staff have a NBS Visit form that cannot be key-entered due to missing or out-of-sequence tasks or if

staff or participants record notes as described in Section 8.2.1.1 - Using the “Notes” Box on the NBS

Forms), then NBS staff should contact the CCC using the steps outlined below:

1. Make a copy of the entire NBS Visit form, Visit 2 (or Visit 4) Participant Update Worksheet, or

the Record Sheet for 24-hour Urine Collection that contains notes or comments.

2. Black out the participant’s name (leave her WHI member ID number).

3. Contact Helen Penor (hpenor @ whi.org) at the CCC by email or phone to let her know that you

are sending a FAX.

4. Fax a copy of the entire NBS Visit form, Visit 2 (or Visit 4) Participant Update Worksheet, or the

Record Sheet for 24-hour Urine Collection to Helen Penor at the CCC.

8.2.2 Management of NBS Forms

During NBS Visits, it is critical that all the pages of NBS forms be kept together. The participant

member ID is only on the first page of the NBS form, thus it is critical that NBS staff not separate the

pages. The form needs travel with the participant.

For example, the 3-hour urine/blood specimens; staff should walk the participant to the urine

collection area and then wait to escort her (and her NBS form) for the 3-hour blood draw.

All completed and key-entered NBS forms (i.e., Form 74 – NBS Screening Result, Form 75 –

NBS Visit 1, etc.) and NBS worksheets (i.e., NBS Visit 1 Eligibility Checklist Worksheet, NBS Visit 2

Participant Update Worksheet, etc.) are placed in the participant’s chart (or special NBS notebook),

even when a participant is ineligible or has declined participation.

For example: Form 74 – NBS Screening Result should be filed in the participant’s chart (or

special NBS notebook) regardless of the screening outcome (i.e., Scheduled, Ineligible or

Declined).

8.3 Data Entry

The NBS data forms are key-entered in a manner similar to any other WHI form, but NBS forms will

have very tight data validation procedures. Staff will not be able to data enter a NBS form if any of the

following situations occur: NBS specimen labels do not match, fields on the form are omitted, or a form

is entered out of sequence. The reason for these validation procedures is that NBS will happen only

once and the timing of the activities is critical. The CCC needs to know that the data is correct, as soon

as possible.

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8.3.1 Trouble Shooting Data Entry

This section provides information to help trouble shoot data entry of NBS Visit forms. For information

about handling NBS Visit forms that cannot be key-entered due to missing or out-of-sequence tasks,

refer to NBS Manual, Section 8.2.1 – Notes on NBS Forms.

8.3.1.1 NBS Labels Do Not Match

If a participant’s NBS label on Form 75 – NBS Visit 1 and Form 76 – NBS Visit 2 are NOT the same

identification number, the form cannot be data entered. If this happens, the CC needs to:

Resolve the problem (if it is an obvious problem) by re-labeling all the woman’s specimens with

a new label set for the two visits (NBS Visit 1 and NBS Visit 2).

Contact the CCC to help work through the problem.

8.3.1.2 Field Omissions

WHILMA will not allow a NBS Visit form (Form 74, 75, 76, 77, 78) to be data entered if any

information is omitted or missing on the form(s). To help prevent potential field omissions and quickly

resolve omissions that do occur, CCs are asked to use the thoroughly review the forms and ensure that

all required NBS tasks are completed.

Completeness Checklist and Completeness Staff IDs:

Form 75 - NBS Visit 1 and Form 77 - NBS Visit 3 have a ‘Completeness Checklist’ that helps staff

review the form. It is important for the NBS-Lead ops (or designee) to review the form and check

to see that all NBS visit activities have been completed and the information has been entered

correctly on the form. The Lead-ops (or designee) reviewing Form 75 or Form 77, enters their

Staff ID on the last page the form, Qx. 20 – Completeness Staff ID to indicate that the form has

been reviewed and checked for completeness.

Form 76 - NBS Visit 2 and Form 78 - NBS Visit 4 do not have a completeness checklist, but do

have a place to record a ‘Visit Completeness Staff ID’. NBS staff who review the form (Form 76

or Form 78) for completeness record their staff ID in Qx. 15 – Visit Completeness Staff ID.

8.3.1.3 Time Sequence of NBS Visit Activities

Spot urines, DLW dosing, meal replacement beverage, and intake of other beverages must be entered in

the sequence specified on the NBS Visit form. WHILMA will not allow a NBS Visit form to be data

entered if expected tasks are out-of-sequence. For example:

A Form 75 – NBS Visit 1 cannot be data entered if the time(s) entered for Qx. 18 – Other

Beverages Consumed are earlier than 2 hours post DLW dosing.

8.3.1.4 Forms Entered Out-of-Sequence

WHILMA will not allow a NBS form to be data-entered out-of-sequence. Checks are in place to

enforce the sequence of NBS forms. For example:

A Form 75 – NBS Visit 1 cannot be data entered unless a Form 74 – NBS Screening Result exists

indicating that the woman had been scheduled.

8.4 NBS WHILMA Upgrade Notes

The CCC provides WHILMA Upgrade Notes for the NBS Recruitment Tracking report and data entry

of NBS forms. NBS WHILMA Upgrade Notes are located in the Outlook Public Folders/All Public

Folders/Nutri Biomarkers Study. CCs are encouraged to print a copy of NBS WHILMA Upgrade Notes

and place them in the NBS Manual, Section 8 – Data Management.

NBS Manual Page 61


SECTION 9

NBS FORMS AND WORKSHEETS

9.0 Overview

This section provides copies of the NBS study forms and worksheets. Accompanying each form is a

detailed set of instructions describing who completes the form, when and how each data item should be

coded, and what should happen to the form when it is completed. Copies of the following NBS forms

and worksheets are included in this section.

9.1 Printing NBS Forms and Worksheets

CCs will receive ‘PDF’ files of all NBS Visit forms (i.e., Forms 74, 75, 76, 77, and 78) and NBS

Worksheets (i.e., NBS Visit 1 Eligibility Checklist Worksheet, NBS Visit 2 Participant Update

Worksheet, NBS Visit 2 (or Visit 4) PABA Worksheet for Staff). Clinics use these electronic files or the

hard copies available in the NBS Manual, Section 9 – NBS Forms and Worksheets to make copies of

forms and worksheets, as need. For information about NBS printed materials, refer to NBS Manual,

Section 2.4.1 – Supplies Obtained by Each CC.

Forms & Worksheets – NBS Visit 1 and NBS Visit 2:

Form 74 – NBS Screening Result

Form 74 Forms Instructions


Form 75 – NBS Visit 1






Forms & Worksheets – NBS Visit 3 and NBS Visit 4 (Reliability Subsample):







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SECTION 10

INDIRECT CALORIMETRY

10.0 Overview of Indirect Calorimetry

The measurement of resting energy expenditure (indirect calorimetry) will be done in a subset of the

clinics participating in the Nutritional Biomarkers Study (Chicago Northwestern, Madison and

Pittsburgh). These clinics will supply their own metabolic cart and train their own staff.

Resting metabolic rate will be measured with a Deltatrac II Respiratory Gas Analyzer, a Sensormedics

29, or similar instrument. These are semi-portable units that measure the concentrations of oxygen and

carbon dioxide in air streams entering and exiting a clear plastic hood placed over the participant’s head.

Oxygen consumption and carbon dioxide production are calculated from the change in concentration

and flow rate. The measurement must be made under standard conditions and requires about 30-40

minutes to complete. A 30-minute rest period is required prior to the start of the indirect calorimetry

test to ensure that resting energy expenditure is measured.

The resting energy expenditure measurements will serve the following purposes:

Identify the physiologic determinants of total energy expenditure

Provide a measure of physical activity by difference when used in conjunction with total energy

expenditure

10.1 Recruitment

Women participating in NBS at the Chicago-Northwestern, Madison and Pittsburgh clinical centers will

be invited. Up to 150 women from all three clinical centers will complete the indirect calorimetry

procedures. No additional women will be recruited exclusively for the indirect calorimetry.

Staff will explain the indirect calorimetry procedures at NBS Visit 1 and those participants who wish to

participate will do so at NBS Visit 2 or within a week of their NBS Visit 2 appointment.

Participants are not obligated to participate in the indirect calorimetry. They may participate in the rest

of the Nutritional Biomarkers Study without completing the indirect calorimetry. Participants taking

part in the indirect calorimetry will not be offered any additional funds to compensate for time and

travel expenses, but they will be offered their resting energy expenditure data (the number of calories

their body burns at rest).

10.2 Eligibility

Women invited to participate in the indirect calorimetry test need to be listed on either the NBS

Recruitment Tracking report (NBS001) or the NBS Supplemental Recruitment Tracking report

(NBS002) for expanded NBS recruitment.

Participants who have self-reported claustrophobia, which would cause them to become anxious when

the plastic RMR hood is placed over their head should be excluded from taking part in the indirect

calorimetry procedures. The procedures require that the participant lie quietly for 30 minutes and

anxiety caused by claustrophobia would produce inaccurate test results.

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10.3 Study Procedures

Clinics may conduct the indirect calorimetry test on the same day as NBS Visit 2 (either pre or post

NBS Visit 2) or on a separate day (within a week of NBS Visit 2). For an overview and description of

NBS Visit 2 activities, refer to NBS Manual, Section 5.2 – NBS Visit 2 (Day 15).

The indirect calorimetry procedures will add approximately 1 to 1½ hours to the total time for NBS

Visit 2 (total time for both NBS Visit 2 and indirect calorimetry would be about 3 hours).

10.3.1 Pre-Visit 2 Reminder Call

When the participant’s NBS Visit 2 and indirect calorimetry test are on the same day, call the participant

two days prior to the participant’s scheduled NBS Visit 2. Confirm the participant’s NBS Visit 2 and

indirect calorimetry appointments.

Remind the participant to:

Fast for 12 hours (instead of 6 hours) prior to her clinic appointment. Ask the participant to stop

eating or drinking anything except water (no caffeinated beverages such as black coffee or tea are

permitted before indirect calorimetry testing) after 8 PM the night before the scheduled clinic

visit for measurement of resting energy expenditure (12 hour fast).

Possible Script: “You are scheduled for a clinic visit at (time and date). During this visit, you will

undergo a test to measure the number of calories that you burn at rest. In order to obtain accurate

test results, you must not eat after 8 PM the night before the visit or on the morning before the

visit. You may not drink any calorie containing beverages on the morning of your visit. It is also

important that you do not smoke or take any nicotine products (chew, nicotine gums or patches),

any caffeine containing products (coffee, tea, or diet sodas, or drugs to stay awake) for 2 hours

before the visit.”

Complete her 24-hour urine collection. Refer to NBS Manual Section 5.2.2.2.1 – Review

Instructions for 24-hour Urine Collection for a list of information to cover.

Take all her regular medications with water.

Wear clothing that allows the sleeve to be easily raised above the elbow without constricting the

blood flow to the forearm and hands and allows for ease of multiple urine collections.

Ask the participant if she has questions and provide information as needed.

10.3.2 Indirect Calorimetry Procedures

The indirect calorimetry measurement may be conducted either before or after NBS Visit 2 procedures,

but the participant must remain fasted until the conclusion of the indirect calorimetry test and until the

NBS Visit 2 fasting blood has been drawn.

Below is an overview of the steps taken to measure energy expenditure (indirect calorimetry):

1. Ensure that the equipment has been properly calibrated at the start of each day.

2. Ensure that the equipment is turned on and warmed up for at least 30 minutes prior to using it

with a participant.

3. Ask the participant to lie down and rest quietly for about 30 minutes.

4. If the participant feels cold, offer her a blanket. If the participant feels hot, alter the environment

to insure that she does not sweat.

5. Check that the monitor is in canopy mode. Change if needed (This may differ between

instruments).

6. Check that the monitor is in the artifact suppression mode with a 10 min start delay (This may

differ between instruments).

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7. Check that the hoses from the hood to the metabolic monitor are connected and the unit is turned

on.

8. Perform a calibration of the metabolic monitor.

9. After the initial 30 minute rest period, measure the resting metabolic rate as per instrument

instructions.

10. The printer should be reporting data on a minute by minute basis. If it is not printing, check the

connections, printer power, or see the PRINTER SETUP.

11. Proceed with the measurement for 30-40 min.

The technician must remain with the participant - monitoring gas flow alarms and visually

checking for labored breathing to insure that gas flow does not fail.

The participant must remain at rest but not sleep.

The participant must not talk, except when necessary to communicate a potential problem. If

the participant does talk, lift their arms to scratch an itch, shift their weight to prevent

stiffness etc, indicate the time and movement on the printout using a pen or pencil.

Confirm that the participant is still thermally comfortable.

If the participant has to get up because she needs to use the bathroom, then the measurement

can be terminated, but the participant will need to start over. The measurement sequence (i.e.,

steps 3-19) needs to be repeated beginning with a 10 min rest in place of the 30 min called for

in the basic protocol.

12. At 30-40 minutes, check the display data printout for a stable reading.

13. End the measurement.

14. Obtain the output data from the metabolic cart.

15. Remove the hood from over the participant’s head.

16. Ask the participant to sit upright.

17. Help the participant to their feet and be sure that they steady. Remember that they have fasted

and there is a small risk of hypoglycemia.

18. Give the participant her snack, if she has completed the NBS Visit 2 fasting blood draw.

19. Offer the participant her resting energy expenditure results. Answer any questions.

Note: The primary safety concern is that airflow through the hood is maintained while the hood is in place

over the participant’s head. Loss of flow due to a rare failure of the fan in the metabolic cart or due to a

loose hose will cause discomfort and in an extreme case may cause asphyxiation. Although an alarm will

sound if the unit does not detect breathing, the EE technician should remain with the participant throughout

the measurement. Care should also be exercised when the participant stands-up after the measurement

should dizziness develop secondary to the fast.

10.3.3 Post- Indirect Calorimetry Quality Check

After completion of the indirect calorimetry test, complete a quality check on the following:

1. The printout/electronic record is legible. If not, correct problem and reprint.

2. The average RQ is between 0.75 and 0.9. Values outside of this range may indicate that the participant

fasted longer than 15 h (<0.75), ate within the last 6 h (>0.93), or hyperventilated during the

measurement (>0.93). Other possible explanations are very high fat diets (<0.75), a weight loss diet

(<0.75) or very high carbohydrate diets (>0.93). If the participant admits to a recent meal, reschedule

the test.

3. Check that the coefficient of variation is less than 10%. Possible explanations are excessive participant

movement, irregular breathing pattern, failure to suppress the first 10 min of the measurement, or

instrument maintenance problems. If the first 10 min of the measurement were not deleted, manually

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calculate the average and SD without the first 10 min. If the revised coefficient of variation is less than

10%, record these values. If not, repeat the measurement of resting metabolic rate.

10.4 Data Management

For data collection and management, CCs conducting indirect calorimetry should use the following steps:

Obtain a printout of the participant’s indirect calorimetry results (minute-by-minute and summary

information).

Place the participant’s “NBS Indirect Calorimetry” label from her NBS label set on the first page

of her indirect calorimetry printout.

Place the participant’s WHI Member ID on each page of her indirect calorimetry printout.

Xerox a hard copy of the printout (minute-by-minute and summary information) and staple.

Place the xeroxed copy of the indirect calorimetry printout in the participant’s NBS chart.

Send the original indirect calorimetry printout to Jill Shupe at the WHI Coordinating Center

(CCC) by Federal Express at the end of each week (Friday). If you have printouts for more than

one participant for that week, send all the printouts in the same Fed-Ex package.

Contact Jill Shupe ([email protected]) each time you send a Fed-Ex package to let her know when

the package containing indirect calorimetry results will arrive.


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Appendix for Section 10

Indirect Calorimetry

Table of Contents

This appendix provides copies of the materials used in the Indirect Calorimetry subsample of the Nutritional Biomarkers

Study.

Contents Page

NBS Letter of Invitation (Ver. 3) ..................................................................................................................................... 67

NBS Consent, (Ver. 3), July 2004 .................................................................................................................................... 69

Study-At-A-Glance (Ver. 3), July 2004 ........................................................................................................................... 75

NBS Manual Page 67

WHI NUTRITIONAL BIOMARKERS STUDY (NBS)

<WHI Letterhead>

WHI Nutritional Biomarkers Study

Letter of Invitation to Participants-Version 3

<Date>

<Name>

<Address>

Dear <Name>,

Thank you for your participation in the Women’s Health Initiative Dietary Study. We are now inviting you to

participate in a new research study from the WHI called the WHI Nutritional Biomarkers Study. This study

will look at better ways to measure dietary intake of calories, protein, vitamins and minerals. It will also help

the WHI scientists better understand the final results from the WHI Dietary Study.

The tests for measuring the nutritional biomarkers are simple. We will ask you to attend two visits at the WHI

clinic. You may start the study any time, but if you are due for an Annual Visit, it may be convenient to begin

on the same day as your next Annual Visit. The second visit will be two weeks later.

At the first visit, you will be given a special kind of water to drink that will allow us to measure how many

calories your body uses during the next two weeks. The special water is tasteless and harmless and contains a

type of oxygen and hydrogen that we can measure in your urine.

You will need to come into the clinic after fasting (no food but you may drink liquids) for four hours, then

stay at the clinic for five hours and give four urine samples. We will also measure your height and weight, ask

you to complete a food frequency questionnaire, questions about any dietary supplements you may use and

questions about your usual physical activity. If you are age 60 years or older, we will take a blood sample

from a vein in your arm

We will ask you to return to the clinic two weeks later. The day before your return we will ask you to collect

all of your urine for 24 hours in a container that we provide for you. You will need to fast for twelve hours

before the second visit and it will last three hours.

During the second visit we will take a blood sample, ask you to give two more urine samples, and measure

your weight. We will also conduct a breath test that will measure how much energy your body uses at rest.

During the breath test we will place a plastic ventilated hood over your head and ask you to lie quietly and

breathe normally for 30 minutes.

We will go over these procedures with you again before the study starts.

You should follow your usual eating and activity habits during the two weeks of the study. You will receive a

complementary meal at the end of each clinic visit. You will receive $100.00 when you complete the study

procedures. You may be invited to repeat the procedures in six months.

You were selected from women who are participating in the WHI Dietary Study. Your name has been used

only for the purpose of contacting you and will not be released to anyone. All the information you provide

will be kept confidential in accordance with Federal laws.

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As usual only your Clinical Center and security-cleared staff at the Clinical Coordinating Center in Seattle,

WA will have access to your personal information. Your personal identity will not be revealed in any

publications or results. Your participation is voluntary and will not affect your participation in the Women’s

Health Initiative.

Our clinic staff will telephone you within one week to discuss your interest in participating. We hope you

will contribute your time to this important study.

Sincerely,

<CC PI>

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CONSENT TO PARTICIPATE IN

THE WOMEN'S HEALTH INITIATIVE (WHI) NUTRITIONAL BIOMARKERS STUDY

WHI Coordinating Center

Fred Hutchinson Cancer Research Center

Seattle, Washington [Clinical Center]

[Principal Investigator]

[24-Hour Contact]

This consent form is to tell you about the Nutritional Biomarkers Study, which is a new study within the Women's Health Initiative (WHI). You are being asked to take part in this study because you are a participant in the WHI Dietary Study. The purpose of this consent form is to give you information you will need to help you decide whether or not you want to be in the new study. Please read the information carefully. We encourage you to ask questions about the purpose of the research, what we would ask you to do, the possible risks and benefits, your rights as a study participant and anything else about the research or this form that is not clear. When all your questions have been answered, you can decide if you want to be in the study or not. This process is called informed consent.

WHY IS THIS STUDY BEING DONE?

This research is being done because we want to understand about biological markers of foods that women eat. These “biomarkers” are levels of nutrients from foods people eat, such as vitamins, minerals, fat, calories and protein that are found in blood and urine. This research will help the WHI scientists better understand the final results from the WHI Dietary Study.

HOW MANY PEOPLE WILL TAKE PART IN THIS STUDY?

We expect that 550 women who are part of the WHI Dietary Study across the country will participate in this Nutritional Biomarkers Study. Women in the Dietary Change group and women in the Comparison Group are being asked to participate in this new study.

WHAT IS INVOLVED IN THE STUDY?

If you decide to participate, we will ask you to come to your WHI Clinical Center in <Insert name of Clinical Center> for two visits, which will be two weeks apart. While you may begin the study any time at your convenience, the first visit may be the same day as your regularly scheduled WHI Annual Visit, if you are currently due for an Annual Visit. This first visit will last about 5 hours and the second visit, two weeks later will last about 3 hours.

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Visit one

Before the first visit we will ask you not to eat food or drink any coffee or tea for four hours before coming to the clinic. You may drink water during these four hours, but coffee or tea may cause dehydration, so we ask that you avoid those beverages during the four hour fast.

During this first clinic visit we will first measure your height and weight and then ask you to drink about ½ cup of a solution called “Doubly Labeled Water.” This special water is tasteless, odorless and colorless. It contains naturally occurring special types of hydrogen and oxygen that we can measure in your urine. Drinking the special water on an empty stomach helps it to be absorbed fast. We will ask you to give four urine samples over a four-hour period (one urine sample per hour). These measurements from your urine tell us how much energy (calories) your body uses over the two-week period of the study. If you are age 60 years or older we will take about 2 teaspoons of blood from a vein in your arm at the first visit, about three hours after you drink the doubly labeled water. For all participants, we will ask you to complete a food questionnaire, a dietary supplement use questionnaire, and questions about your usual physical activity. These are questionnaires that you have already completed during other WHI Annual Visits, but they are important to complete again as part of the Nutritional Biomarkers Study. If you are completing your WHI Annual Visit at the same time as the Nutritional Biomarkers Study, you will also complete any of the usual tasks that are part of your WHI Annual Visit during this first visit. For example, this may include a clinical breast exam or a review of medications you may be taking. You may complete the questionnaires and the other Annual Visit tasks in between the urine collections, if necessary.

We will give you a meal replacement beverage (like “Boost” or “Sustacal”) one hour after drinking the special doubly labeled water solution and you will receive a complementary meal after you give the fourth urine specimen.

At home between clinic visits

At the end of the first visit we will give you a urine collection kit and explain to you how to collect all of your urine at home for a 24-hour period. You should collect your urine the day before you return for the second visit. You should keep the urine you collect in special bottles we will provide for you and keep them in the refrigerator until you come back to the clinic. You may be asked to take a vitamin pill the day of the 24-hour urine collection. The vitamin should be taken with each meal on the day you collect your urine. This vitamin is called “para amino benzoic acid,” or PABA. It is a B-vitamin and is excreted completely in the urine. It will help us know that the all excreted urine has been collected.

Visit Two

Before the second visit we will ask you not to eat anything for twelve hours before coming to the clinic. You may drink water, but no coffee or tea. At the second visit you will bring the urine you collected back to the clinic. Our trained staff will take about two tablespoons of blood from a vein in your arm. Fasting for 12 hours before the blood draw helps us to get

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test. We will measure your weight and ask you to give two more urine samples in the clinic, each about one hour apart. The blood samples and urine will be used to measure energy, protein, fats, vitamins and minerals. We will give you a complementary meal or snack when you are done with the tests at the second visit.

During the second visit we will also perform a breath test to measure how much energy your body uses. During the breath test, we will place a ventilated plastic hood over your face and we will ask you to lie quietly for 30 minutes. You will breathe normally during the breath test.

You will receive $100.00 at the end of the second visit for time and travel expenses.

We cannot provide the $100.00 to participants who do not return for the second clinic visit procedures.

If you complete the breath test we will tell you how many calories your body uses each day.

Your participation in this Nutritional Biomarkers Study will be completed at the end of the second visit. However, we are asking 110 women to repeat the study in six months to help us learn how reliable the energy and blood tests are. Participants who repeat the entire study will also complete two 20 minute dietary interviews, one within three weeks after completing the study the first time and the second within about three weeks of the study the second time at the six month point. Trained staff will call you and ask you about all the foods and beverages you consumed the previous day. The dietary interview may sound familiar, as you may have received similar telephone interviews as a participant in the WHI.

We will ask you at the end of the second visit if you want to repeat the study procedures in six months. If you repeat the study you will receive an additional $100.00.

Participation in the Nutritional Biomarkers Study does not affect your participation in the WHI Dietary Study or any other part of WHI.

HOW LONG WILL I BE IN THE STUDY?

You will be in the Nutritional Biomarkers Study for two weeks. You may stop participating at any time.

If you choose to repeat the study in six months, your participation will last again for two weeks.

You will remain a participant in the WHI Dietary Study until it is over in 2005.

NBS Manual Page 72


WHAT ARE THE RISKS OF THE STUDY?

You may feel dizzy for a few minutes after drinking the special doubly labeled water. To prevent you from feeling dizzy we will ask you to remain seated for about 15-20 minutes after drinking it. Blood will be taken from a vein in your arm. This may cause discomfort and a bruise at the site of the needle puncture. All efforts will be made to minimize this risk. People with known allergies to PABA may get a slight rash if they take the PABA vitamin. If you know you are allergic to PABA, we will not ask you to take it. You may find it inconvenient to collect urine for a 24-hour period. If you are claustrophobic or do not like being in small spaces, we will not conduct the breath test if you think you would feel uncomfortable with these procedures. We do not expect there to be any medical problems as a result of your participation in this study. If medical problems do occur, we will refer you to your personal physician, or for other appropriate care. You or your health insurance will be responsible for the costs of any such care.

ARE THERE BENEFITS TO TAKING PART IN THIS STUDY?

We hope the information learned from this study will benefit the WHI Dietary Study. The knowledge gained may help scientists learn about blood nutrients and energy needs in postmenopausal women.

WHAT OTHER OPTIONS ARE THERE? You may choose not to participate in this study.

WHAT ABOUT CONFIDENTIALITY?

All efforts will be made to keep your personal information confidential. We cannot guarantee absolute confidentiality. Your personal information may be disclosed if required by law.

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The Office for Human Research Protection and the Institutional Review Board at <insert name of local CC> may have access to participant research records to ensure that participant rights are being met. We will access your other research records within the Women’s Health Initiative, such as Food Questionnaires you have completed in the past and information about your age and race/ethnicity. We will only access personal health information that is part of data that have been collected for the Women’s Health Initiative. We will not request any personal health information from other sources, such as hospitals or your physician for the Nutritional Biomarkers Study. The information from this study may be published in scientific journals or presented at scientific meetings but your identity will be kept strictly confidential.

WHAT ARE THE COSTS?

Taking part in this study may lead to costs to you or your insurance company. Please ask about any expected costs or insurance problems. In the case of injury or illness resulting from this study, emergency medical treatment is available but will be provided at the usual charges. No funds have been set aside to compensate you in the event of injury. You or your insurance company will be charged for medical care and/or hospitalization.

WHAT ARE MY RIGHTS AS A PARTICIPANT? Taking part in this study is voluntary. You may choose not to take part or you may leave the study at any time. Leaving the study will not result in any penalty or loss of benefits to which you are entitled. If you decide to leave the Nutritional Biomarkers Study it will not affect your participation in the WHI Dietary Study.

WHOM DO I CALL IF I HAVE QUESTIONS? For questions about the study or a research related injury, contact the WHI Clinical Center PI, <name> at <phone number> or any of the investigators listed at the beginning of this form. For questions about your rights as a research participant, contact the <name of Center> Institutional Review Board (which is a group of people who review the research to protect your rights) at <telephone number>.

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INVESTIGATOR'S STATEMENT My signature below indicates that I have provided an explanation of the above research program. I have explained the risks and benefits of this study and the participant was given an opportunity to ask questions and have them answered. The participant was given an opportunity to discuss the procedures, including possible alternatives and risks, and to ask any additional questions. A signed and dated copy of the consent form has been given to the participant. ____________________________________ _______________ Signature of Principal Investigator or Designated Study Staff Date PARTICIPANT STATEMENT I have read or had read to me the information provided about this study and have had the opportunity to ask questions about this study. These questions have been answered to my satisfaction. I voluntarily consent to join this new study in the Women’s Health Initiative. I understand that any questions I may have about this research in the future will be answered by one of the investigators listed on this form, and that any questions I have about my rights as a research participant will be answered by the persons listed above. I am aware that I and/or my insurance carrier is responsible for any medical costs that may arise from my participation in this research study, including adverse effects. My signature below acknowledges that I voluntarily consent to participate in this study and that have received a signed copy of this consent form. ___________________________________ _______________ Signature of Participant Date ___________________________________ _______________ Signature of Witness Date Copies to: Participant

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<WHI Letterhead>

WHI Nutritional Biomarkers “Study At A Glance” (Ver. 3)

Thank you for your interest in the WHI Nutritional Biomarkers Study.

Here is what we will ask you to do over the two weeks of the study.

Visit 1 (Day 1)

Come to the WHI Clinical Center having fasted (no food) for four hours. Please bring your dietary

supplement bottles.

While fasting, at the visit:

We will measure your height and weight

You will give a urine sample

You will drink the special water solution for measuring the calories you use

One hour later:

You will be given 8 ounces of a liquid meal replacement to drink (Sustacal or similar)

Two through five hours later:

You will give a urine sample at about 2, 3 and 4 hours after drinking the special water solution –

we will tell you when you need to give the urine samples.

If you are age 60 years or older, we will take a small (2 tsp) blood sample 3 hours after you drink

the special water

You will complete a food questionnaire, a dietary supplement questionnaire, and questions about

your usual physical activity

At the end of the first visit:

You will receive instructions for the second visit.

You will receive a complementary meal

At home (Day 14)

Collect all of your urine for the 24-hour period in the special containers we give you. Keep the

urine in the refrigerator.

Take the Special B-Vitamin if asked to do so

Visit 2 (Day 15)

Come to the WHI clinic having fasted (no food) for 12 hours. Bring the urine you collected.

While fasting, at the visit:

We will measure your weight

We will take a sample of blood (about 2 Tbsp) from a vein in your arm

You will give two urine samples – one hour apart – we will tell you when you need to give the

urine samples

You will complete a breath test – we will place a plastic ventilated hood over your head and ask

you to lie quietly for about 30 minutes.

Two to three hours later, at the end of the visit:

You will receive a complementary meal

You will receive $100.00 for your time and travel

You will receive information on the number of calories your body uses each day

Please ask the clinic staff if you have any questions

Thank you for your participation in this important study!

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WHI NUTRITIONAL BIOMARKERS STUDY OVERVIEW ....................................................................1

1.0 Overview of the WHI Nutritional Biomarkers Study (NBS) ............................................................1

GUIDELINES ....................................................................................................................................................2

2.0 Overview ................................................................................................................................................2 2.1 Staffing ...................................................................................................................................................2 2.1.1 NBS Lead-Ops .......................................................................................................................................2 2.2 Training and Certification ...................................................................................................................2 2.2.1 NBS Lead-Ops Person ..........................................................................................................................2 2.2.2 Non-Lead NBS Staff .............................................................................................................................3 2.2.3 Training Resources ...............................................................................................................................3 2.2.3.1 NBS Training Conference Calls ..........................................................................................................3 2.2.3.2 NBS Procedure Manual (referred to as the NBS Manual) ................................................................3 2.3 Communication .....................................................................................................................................3 2.3.1 Email Communication ..........................................................................................................................3 2.3.2 WHI Public Folders ..............................................................................................................................4 2.4 Supplies ..................................................................................................................................................4 2.4.1 Supplies Obtained by Each CC ...........................................................................................................4 2.4.2 Supplies Provided by the CCC ............................................................................................................5 2.4.3 Supplies Provided by Dale Schoeller (University of Wisconsin) .......................................................5 2.4.4 Guidelines for Ordering Supplies ........................................................................................................6

RECRUITMENT .............................................................................................................................................10

3.0 Overview ..............................................................................................................................................10 3.1 NBS Study Sample ..............................................................................................................................10 3.2 Recruitment Goals ..............................................................................................................................10 3.3 Recruitment Plan ................................................................................................................................10 3.4 Recruitment List .................................................................................................................................11 3.5 Invitation Letter ...................................................................................................................................11 3.6 WHILMA Resources for NBS Recruitment Tracking .....................................................................12 3.6.1 NBS Recruitment Tracking Report ...................................................................................................12 3.6.2 NBS Custom Data Extract ..................................................................................................................12 3.7 Expanded NBS Recruitment (Beginning July 2004) ........................................................................12 3.7.1 Expanded Recruitment Plan ...............................................................................................................12 3.7.2 NBS Study Materials for Expanded Recruitment ............................................................................12 3.7.3 Supplemental Recruitment List for Expanded Recruitment ...........................................................12 3.7.4 Approaching Participants for Expanded Recruitment ....................................................................12

SCREENING ....................................................................................................................................................13

4.0 Overview of Screening Process ..........................................................................................................13 4.1 Screening Activities .............................................................................................................................13 4.1.1 Telephone Contact ..............................................................................................................................13 4.1.2 Determine Eligibility and Interest ......................................................................................................13 4.1.3 Schedule NBS Visit 1 ...........................................................................................................................13

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4.1.4 Complete and Data Enter NBS Screening Result (Form 74) ...........................................................14

NBS VISITS ......................................................................................................................................................15

5.0 Overview of NBS Visits ......................................................................................................................15 5.1 NBS Visit 1 (Day 1) .............................................................................................................................15 5.1.1 Overview of Activities .........................................................................................................................15 5.1.2 Integration of NBS Visit 1 and Annual Visit .....................................................................................17 5.1.3 Pre-NBS Visit 1 Activities ...................................................................................................................21 5.1.3.1 Supplies and Forms ...............................................................................................................................21 5.1.3.2 Pre-Visit 1 Reminder Call .....................................................................................................................21 5.1.4 NBS Visit 1 Activities ..........................................................................................................................23 5.1.4.1 Reception ..............................................................................................................................................23 5.1.4.2 Eligibility ..............................................................................................................................................23 5.1.4.3 NBS Consent .........................................................................................................................................23 5.1.4.4 Provide Study at a Glance Handout ......................................................................................................24 5.1.4.5 Physical Measurements (height and weight) ........................................................................................25 5.1.4.6 Spot Urine Collection – NBS Visit 1 ....................................................................................................25 5.1.4.6.1 General Information ........................................................................................................................25 5.1.4.6.2 Time 0 (Fasting) Urine Collection ...................................................................................................25 5.1.4.7 Administration of Doubly Labeled Water (DLW) – Fasting ................................................................26 5.1.4.7.1 DLW Doses .....................................................................................................................................26 5.1.4.7.2 DLW Administration .......................................................................................................................26 5.1.4.7.3 Handling DLW Spillage ..................................................................................................................26 5.1.4.7.4 Storage of DLW ...............................................................................................................................27 5.1.4.8 Provide WHI Forms 35 & 60 and Complete Task 45 ...........................................................................27 5.1.4.9 Provide Meal Replacement Beverage (MRB) ......................................................................................27 5.1.4.10 Two Hour Urine Collection ..................................................................................................................27 5.1.4.10.1 Handling Collection Problems .......................................................................................................28 5.1.4.11 Three Hour Urine Collection ................................................................................................................28 5.1.4.12 Three Hour Blood Draw (for women > 60) ..........................................................................................28 5.1.4.13 Collect and Review Form 35 and Form 60 ...........................................................................................29 5.1.4.14 Four Hour Urine Collection ..................................................................................................................29 5.1.4.15 Other Beverages ....................................................................................................................................29 5.1.4.16 Explain and Provide Materials for 24-hour Urine Collection ...............................................................30 5.1.4.17 Confirm Appointment for NBS Visit 2 .................................................................................................31 5.1.4.18 Provide Complementary Meal ..............................................................................................................31 5.1.4.19 Check Visit 1 Completeness .................................................................................................................31 5.1.5 Post-NBS Visit 1 Activities ..................................................................................................................32 5.1.5.1 Follow-up on Missing Forms ................................................................................................................32 5.1.5.2 Data Entry .............................................................................................................................................32 5.2 NBS Visit 2 (Day 15) ...........................................................................................................................33 5.2.1 Overview of Activities..........................................................................................................................33 5.2.2 Pre-NBS Visit 2 Activities ...................................................................................................................34 5.2.2.1 Supplies and Forms ...............................................................................................................................34 5.2.2.2 Pre-Visit 2 Reminder Call .....................................................................................................................34 5.2.2.2.1 Review Instructions for 24-hour Urine Collection (Day 14) ...........................................................34 5.2.2.2.2 Confirm Appointment and Fasting Time .........................................................................................35 5.2.3 NBS Visit 2 Activities ..........................................................................................................................35 5.2.3.1 Reception – Welcome ...........................................................................................................................35 5.2.3.2 Complete NBS Visit 2 Participant Update Worksheet ..........................................................................36 5.2.3.3 Receive 24-hour Urine Collection ........................................................................................................36

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5.2.3.4 Physical Measurements (weight) ..........................................................................................................37 5.2.3.5 Spot Urine Collection - NBS Visit 2 .....................................................................................................37 5.2.3.5.1 General Information ........................................................................................................................37 5.2.3.5.2 Time 0 (Fasting) Urine Collection ...................................................................................................37 5.2.3.6 Fasting Blood ........................................................................................................................................37 Be sure to protect all the blood samples collected at NBS Visit 2 from natural and fluorescent light. For

example, cover vacutainers with foil until processing is completed (refer to NBS Manual, Section 7.3.3.2.3 –

Time 0 (Fasting) Blood Draw). ..........................................................................................................................37 5.2.3.6.1 Handling Non-Fasting Participants..................................................................................................37 5.2.3.7 Snack/Meal ...........................................................................................................................................37 5.2.3.8 One Hour Urine Collection ...................................................................................................................37 5.2.3.9 Invite Repeat of NBS - (Reliability Subsample) ...................................................................................37 5.2.3.10 Make Arrangements for Time and Travel Costs and Provide Thank You for Participation ................37 5.2.4 Post-NBS Visit 2 Activities ..................................................................................................................37 5.2.4.1 Data Entry .............................................................................................................................................37 5.2.4.2 24-hour Recalls .....................................................................................................................................37

NBS RELIABILITY SUBSAMPLE (INFORMATION TO BE ADDED) ...............................................38

BLOOD AND URINE COLLECTION, PROCESSING, AND SHIPMENT .............................................39

7.1 General Guidelines..............................................................................................................................40 7.1.1 Safety Procedures - Precautions for Handling Blood .......................................................................40 7.1.2 Safety Procedures - Precautions for Handling Urine .......................................................................40 7.1.3 Training and Certification ..................................................................................................................40 7.1.4 Facilities ................................................................................................................................................40 7.1.5 Supplies .................................................................................................................................................40 7.1.6 Labels ....................................................................................................................................................40 7.1.7 Quality Control (QC) for Laboratory Analysis ................................................................................44 7.2 NBS Visit 1 (or NBS Visit 3) ...............................................................................................................45 7.2.1 General Blood and Urine Sampling and Aliquot Schedule and Quality Control (QC) ................45 7.2.1.1 Sampling and Aliquot Schedule ............................................................................................................45 7.2.1.2 Quality Control (QC) Samples for NBS Visit 1 (or NBS Visit 3) - QC CCs only ...............................45 7.2.2 Day of NBS Visit 1 (or NBS Visit 3) (Day 1) ......................................................................................46 7.2.2.1 Before the Participant Arrives ..............................................................................................................46 7.2.2.2 During the Visit (Overview) .................................................................................................................46 7.2.2.2.1 Timing of the Spot Urine Collections ..............................................................................................46 7.2.2.2.2 DLW Spot Urine Collection Steps ..................................................................................................47 7.2.2.2.3 Time 0 (Fasting) Urine Collection ...................................................................................................47 7.2.2.2.4 Time 2 Urine Collection (2 hours after DLW ingestion) .................................................................47 7.2.2.2.5 Time 3 Urine Collection (3 hours after DLW ingestion) .................................................................47 7.2.2.2.6 Time 3 Blood Draw (3 hours after DLW ingestion, for women 60 years of age or older) .............48 7.2.2.2.7 Time 4 Urine Collection (4 hours after DLW ingestion) .................................................................48 7.2.2.2.8 Instructions for the 24-hour Urine Collection..................................................................................48 7.3 NBS Visit 2 (or NBS Visit 4) (Day 15) ...............................................................................................50 7.3.1 General Blood and Urine Sampling and Aliquot Schedule and Quality Control (NBS-modified)

50 7.3.1.1 Sampling and Aliquot Schedule ............................................................................................................50 7.3.1.2 Quality Control (QC) Samples for NBS Visit 2 (or NBS Visit 4) - QC CCs only ...............................50 7.3.2 One Day Before NBS Visit 2 (or NBS Visit 4) (Day 14) ....................................................................51

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Refer to NBS Manual, Section 5.2.2.2 - Pre-Visit 2 Reminder Call and Section 5.222.1 – Review Instructions

for 24-hour Urine Collection (Day 14) ..............................................................................................................51 7.3.3 Day of NBS Visit 2 (or NBS Visit 4) (Day 15) ....................................................................................51 7.3.3.1 Before the Participant Arrives ..............................................................................................................51 7.3.3.2 During the Visit (Overview) .................................................................................................................51 7.3.3.2.1 Receive the 24-hour Urine Collection From Participant .................................................................52 7.3.3.2.2 Time 0 (Fasting) Urine Collection ...................................................................................................52 7.3.3.2.3 Time 0 (Fasting) Blood Draw ..........................................................................................................53 7.3.3.2.4 Time 1 Urine Collection (1 hour after Time 0 [fasting] urine collection) .......................................53 7.3.3.2.5 24-hour Urine Collection Processing ...............................................................................................53 7.4 Blood Draw Specifics ..........................................................................................................................54 7.4.1 Preparation of the Blood Drawing and Processing Areas for All NBS Visits ................................54 7.4.2 Preparation for the Participants ........................................................................................................54 7.4.3 Venipuncture ........................................................................................................................................55 7.4.4 Blood Collection Problems ..................................................................................................................55 7.4.4.1 Directions for Staff When Participants Are Not Fasted ........................................................................55 7.4.4.2 Handling Participants Who Are Extremely Apprehensive About Having Blood Drawn .....................55 7.4.4.3 Special Consideration for Drawing Blood in the Elderly .....................................................................55 7.4.4.4 Difficult-to-Identify Venipuncture Sites ...............................................................................................55 7.4.4.5 Difficult Venipuncture ..........................................................................................................................56 7.4.4.6 Fainting .................................................................................................................................................56 7.4.4.7 Deficient Serum and Plasma Samples ..................................................................................................56 7.5 Blood Processing .................................................................................................................................56 7.5.1 Blood Sample Handling and Processing: Timelimits .......................................................................56 7.5.2 Operating the Refrigerated Centrifuge .............................................................................................57 7.5.3 Processing Blood Samples ...................................................................................................................57 7.5.3.1 Preparation of Blood Cryovials ............................................................................................................57 7.5.4 Freezing and Storing Blood and Urine Cryovials .............................................................................57 7.5.5 Equipment Quality Assurance............................................................................................................57 7.6 Blood Storage and Shipping ...............................................................................................................57 7.6.1 Shipping Schedule ................................................................................................................................57 7.6.2 Mailing Instructions ............................................................................................................................51 7.6.3 Packaging Instructions ........................................................................................................................51 7.6.4 Receipt of Frozen Specimen Shipment at McKesson BioServices ...................................................51

DATA MANAGEMENT .................................................................................................................................58

8.0 Introduction .........................................................................................................................................58 8.1 NBS Label Set ......................................................................................................................................58 8.2 NBS Data Collection Forms ...............................................................................................................58 8.2.1 Notes on NBS Forms .............................................................................................................................58 8.2.1.1 Using the “Notes” Box’ on the NBS Forms .........................................................................................58 8.2.1.2 Handling Notes on NBS Forms ............................................................................................................58 8.2.2 Management of NBS Forms ................................................................................................................59 8.3 Data Entry ...........................................................................................................................................59 8.3.1 Trouble Shooting Data Entry .............................................................................................................60 8.3.1.1 NBS Labels Do Not Match ...................................................................................................................60 8.3.1.2 Field Omissions ....................................................................................................................................60 8.3.1.3 Time Sequence of NBS Visit Activities ...............................................................................................60 8.3.1.4 Forms Entered Out-of-Sequence ...........................................................................................................60 8.4 NBS WHILMA Upgrade Notes .........................................................................................................60

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NBS FORMS AND WORKSHEETS .............................................................................................................61

9.0 Overview ..............................................................................................................................................61 9.1 Printing NBS Forms and Worksheets ...............................................................................................61

INDIRECT CALORIMETRY ........................................................................................................................62

10.0 Overview of Indirect Calorimetry .....................................................................................................62 10.1 Recruitment .........................................................................................................................................62 10.2 Eligibility ..............................................................................................................................................62 10.3 Study Procedures ................................................................................................................................63 10.3.1 Pre-Visit 2 Reminder Call ...................................................................................................................63 10.3.2 Indirect Calorimetry Procedures .......................................................................................................63 10.3.3 Post- Indirect Calorimetry Quality Check ........................................................................................64 10.4 Data Management ...............................................................................................................................65

THANK YOU FOR YOUR INTEREST IN THE WHI NUTRITIONAL BIOMARKERS STUDY. .....75

HERE IS WHAT WE WILL ASK YOU TO DO OVER THE TWO WEEKS OF THE STUDY. ..........75

Visit 1 (Day 1) ...................................................................................................................................................75 At home (Day 14) .............................................................................................................................................75 Visit 2 (Day 15) .................................................................................................................................................75

PLEASE ASK THE CLINIC STAFF IF YOU HAVE ANY QUESTIONS ...............................................75

Thank you for your participation in this important study! .........................................................................75

Questionnaire/Script Form 74 before AV NBS Telephone ... · Participant Materials and Appendix D...

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Transcript of Questionnaire/Script Form 74 before AV NBS Telephone ... · Participant Materials and Appendix D...